Gene therapy for retinitis pigmentosa caused by MFRP mutations: human phenotype and preliminary proof of concept

Abstract

Autosomal recessive retinitis pigmentosa (RP), a heterogeneous group of degenerations of the retina, can be due to mutations in the MFRP (membrane-type frizzled-related protein) gene. A patient with RP with MFRP mutations, one of which is novel and the first splice site mutation reported, was characterized by noninvasive retinal and visual studies. The phenotype, albeit complex, suggested that this retinal degeneration may be a candidate for gene-based therapy. Proof-of-concept studies were performed in the rd6 Mfrp mutant mouse model. The fast-acting tyrosine-capsid mutant AAV8 (Y733F) vector containing the small chicken β-actin promoter driving the wild-type mouse Mfrp gene was used. Subretinal vector delivery on postnatal day 14 prevented retinal degeneration. Treatment rescued rod and cone photoreceptors, as assessed by electroretinography and retinal histology at 2 months of age. This AAV-mediated gene delivery also resulted in robust MFRP expression predominantly in its normal location within the retinal pigment epithelium apical membrane and its microvilli. The clinical features of MFRP-RP and our preliminary data indicating a response to gene therapy in the rd6 mouse suggest that this form of RP is a potential target for gene-based therapy.

FIG. 1.

Human MFRP-RP phenotype. (A) En face imaging. Digitally stitched wide-field, near-infrared reflectance (NIR-REF) imaging of the patient (top). Arrows point to bone spicule-like pigment. Melanin abnormalities are visible on reduced-illuminance autofluorescence imaging (RAFI) with NIR light (bottom left) and lipofuscin abnormalities are demonstrated on RAFI with short-wavelength (SW) light (bottom right). Insets: Representative normal images for each modality. Images are individually contrast-stretched for visibility of features. (B) Electroretinography. Standard full-field elecroretinograms (ERGs) from a normal subject and a patient with MFRP-RP. Rod b-waves were reduced to 4% of mean normal; mixed ERGs had reduced a-waves (11% of normal) and b-waves (7% of normal); and cone ERGs (1 and 30 Hz) were reduced to 35% and 10% of normal, respectively. Stimulus onset was at the start of traces. Calibrations are shown to the right and below the waveforms. (C) Psychophysics. Dark- and light-adapted static threshold perimetry results are displayed as grayscale maps of rod and cone sensitivity loss. The physiological blind spot is shown as a black square at 12° in the temporal field. N, T, I, and S, nasal, temporal, inferior, and superior visual field, respectively. Kinetic perimetry results (inset, top right) illustrate some visual field constriction (nasally) for the larger target (V-4e) with 70% of normal extent, and a central island with 10% normal extent, using the small target (I-4e). (D and E) Thickness topography of total retina, inner retina, and outer nuclear layer mapped in the central retina for normal subjects (D, n=6; age, 21–41 years) and the patient with MFRP-RP (E). Insets in the lower right-hand corner of patient data indicate whether the thickness measurements are within normal limits (white), abnormally thin (blue, less than 2 SD), or abnormally thickened (pink, greater than 2 SD). (F–H) Retinal laminar architecture. Cross-sectional optical coherence tomography (OCT) images along the horizontal meridian through the fovea in a normal subject (F) are compared with those of a patient with MFRP-RP (G). Examples of OCT cross-sections in other retinopathies with abnormal foveal shapes are shown along with measurements of the hyperreflectivity at the vitreoretinal interface in normal subjects; patients with RP with cystoid macular edema (CME) and choroideremia (CHM); and the patient with MFRP-RP (H). Error bars, 1 SD. MFRP-RP, MFRP (membrane-type frizzled-related protein) gene mutation-associated retinitis pigmentosa.

FIG. 2.

Functional and structural consequences of gene therapy in rd6 mice. (A) Representative ERG traces from a wild-type (WT) mouse eye compared with those from the two eyes of a 2-month-old rd6 mouse that was treated uniocularly with vector–gene 6 weeks previously. Rod-isolated responses were elicited under dark-adapted conditions with dim (0.02 scot-cd·s·m^–2) flashes; mixed rod- and cone-driven responses were elicited with brighter (2 scot-cd·s·m^–2) flashes. Cone-isolated responses were evoked with 25 phot-cd·s·m^–2 stimuli on a rod-desensitizing background. (B) Interocular difference of four ERG parameters plotted for individual rd6 animals (red circles) and wild-type mice (black squares). *p<0.01 for t tests comparing the means of two groups of animals. L-R, left−right; T-U, treated−untreated. (C) Light microscopy of a 2-month-old wild-type retina (left) and untreated (middle) and treated rd6 retinas (right). Note the shorter outer segments (OS), reduced outer nuclear layer (ONL) thickness, and the presence of phagocytic-like cells (arrows) in the untreated rd6 eye. RPE, retinal pigment epithelium; IS, inner segments; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 20 μm.

FIG. 3.

Immunolocalization of MFRP and Western blot analysis in rd6 mice with and without gene therapy. (A) Detection of MFRP expression in wild-type albino control retinas (scale bar, 20 μm). Note the increased distance between the outer nuclear layer (ONL) and retinal pigment epithelium (RPE) [compared with (B) and (C)] caused by artifactual postmortem retinal detachment. Inset: Image of the entire retina at lower magnification (scale bar, 0.5 mm). (B) Untreated rd6 retina showing no detectable MFRP expression. (C) Vector-treated rd6 retina section showing robust MFRP immunostaining predominantly in the RPE apical membrane. Inset: Full retinal section at low magnification, showing the widespread area of transduction (scale bar, 0.5 mm). DAPI (blue) staining of nuclei. (D and E) Higher magnification of the RPE layer from (A) and (C), respectively, depicting strong MFRP expression in the apical RPE membrane and its microvilli (arrowhead) (scale bars, 10 μm). (F) Western blot analysis of whole eyecup protein extracts from a 2-month-old untreated (U) rd6 eye and the partner vector-treated (T) eye. Note the presence of an immunoreactive MFRP band in the treated eye only.