Retinitis pigmentosa induced by rhodopsin mutation
Eye and vision
Vision is one of our most important senses, allowing us to move in space, to distinguish faces and objects, and perceive the world around us. Photons constitute the light stimulus that are detected by our eyes, caught by the retina, transformed to an electrochemical signal and transmitted to the brain for its interpretation. From the visible part of the eye to its deepest structure (Figure 1), the light penetrates into the eye through the cornea, a clear and curved layer that protects it from physical damage and bacterial aggression. Then, the amount of light penetrating the eye is modulated by the dilatation or the constriction of the pupil, the central opening of the iris, which depends on the action of the papillary sphincter muscle and dilatator muscle respectively. The iris thus acts as a diaphragm and gives the color to the eye. The light then runs through the lens that acts like a thin biconvex capsule, able to change its shape through the action of the ciliary muscles, to focus the light onto the retina. Behind the lens stands the vitreous, a viscous transparent substance that participates in the maintenance of the intra-ocular pressure. Finally, photons are caught by the retina, the neuro-sensorial part of the eye. After signal transformation and processing by nerve impulses into the retina (described below) visual information is interpreted by the visual cortex, after its transit through the lateral geniculate nucleus (Figure 2). Due to the neuronal nature of most retinal cells and the presence of structures similar to those found in the brain such as the blood brain barrier, the eye is considered an extension of the brain, and a part of the central nervous system.
The neuroretina, composed of photoreceptors, horizontal cells, bipolar cells, amacrine cells and ganglion cells (Figure 3), is the essential tissue for the capture and transmission of the visual information. When photons enter into the eye, they pass through the retina and are captured by rod and cone photoreceptors, which transform them to an electrochemical signal, itself transmitted to bipolar cells. These last cells extend the signal transmission to ganglion cells, whose fibers form the optic nerve that drives the information into the visual cortex. Amacrine cells and horizontal cells participate in visual information processing by permitting When a photon enters into the eye, it goes across the cornea, the aqueous humor, the pupil, the lens and the vitreous before reaching the retina. Adapted from virtualmedicalcentre.com.
A visual signal reaches the primary visual cortex from the eye, through the optic nerve, the optic chiasm, the optic tract, the lateral geniculate nucleus and the optic radiation.
a better propagation of the signal and improve contrast and definition of the visual stimuli, for example by lateral inhibition.
The neuroretina would not be efficient without the presence of Müller cells, astrocytes and microglial cells, which represent the glial cells of the retina and contribute to maintaining the structure of this tissue. Last but not least of the retinal cells are those of the retinal pigment epithelium, which have many important functions, including recycling of 11-cis-retinal, absorption of scattered light, providing photoreceptors with nutrients and oxygen, and phagocytosis of damaged photoreceptor outer segments.
All these cells are organized in strata, the retina can be divided in 10 layers (Figure 3), from the vitreous to the choroid (i.e. from the inner to the outer retina):
∀ The inner limiting membrane, a basal membrane composed of proteins from the extracellular matrix and feet of Müller glial cells,
∀ The optic nerve fiber layer, constituted of the axons of ganglion cells, which gather to form the optic nerve,
∀ The ganglion cell layer, represented by the cell body of ganglion cells,
∀ The inner plexiform layer, which is an entanglement of axons and dendrites of ganglion, amacrine and bipolar cells,
∀ The inner nuclear layer, which contains the cell bodies of bipolar, amacrine, horizontal and Müller cells,
∀ The outer plexiform layer, composed of axons and dendrites of bipolar and horizontal cells and photoreceptors,
∀ The outer nuclear layer (ONL), formed by the nuclei of photoreceptors (PR),
∀ The outer limiting membrane, which is a “zonula adherens”, a region of adherence between two cell types, here between PR and Müller glial cells,
∀ The segments of the PR, constituted of inner and outer segments (IS-OS),
∀ The retinal pigment epithelium (RPE) composed of cells of the same name.
Two types of PR are present in the retina: cones and rods, which take their names from their shapes (Figure 4). They are made up of (i) a synaptic terminal connected to bipolar and horizontal cells, (ii) a cell body that contains the nucleus, (iii) an IS where metabolism, biosynthesis and endocytosis take place, explaining the presence of numerous mitochondria at this level, (iv) a connecting cilium, the bridge between segments, which shares morphology and composition of a primary cilia, and (v) an OS, composed of around 1000 flattened, lamellar-shaped membrane discs38, which considerably increase the total membrane surface of the cell. These very specific structures mainly contain opsins, light-sensitive molecules that are involved in the first step of the phototransduction cascade that transforms the light stimulus in an electrochemical signal.
Cones and rods are both distributed quite uniformly on the surface of the retina, except in one particular region specific to humans and other higher primates, the macula. The macula is localized in the central retina, where cones are the majority PR. The central part of the macula is the fovea and is exclusively composed of cones (Figure 5), it represents the region where the acuity is maximal. Cones are thus essential to visual acuity, but require normal or high-light conditions. In humans and some primates, each cone synthesizes one of the three cone opsins that possess maximal absorption at different wavelengths of light: (i) long wavelength sensitive opsin (OPN1LW) detects photons in the red region of the visible light spectrum, (ii) middle wavelength sensitive opsin (OPN1MW) for the green region of the spectrum, (iii) and short wavelength sensitive opsin (OPN1SW) for the blue region of the spectrum. These three kinds of proteins allow color vision, which is totally created by the brain, after combinatorial analysis of the input sent by these cells.
In the other hand, rods are implicated in dim-light, and peripheral vision. They are the most numerous cells in our retina, counting for around 60 million per retina, nearly twenty times more represented than cones39. Rods contain only one kind of opsin, called rhodopsin, which represent more than 90% of rod OS proteins and absorbs light maximally at 495nm. While cones continue to function at more than one hundred million photons per second, rods saturate at several thousand photons, but the high density of rhodopsin combined with its sensitivity allows the retina to detect a single photon1. This important difference between rods and cones concerning the amount of light required to induce a response, allows to the retina to cover a large range of light intensities. Rods are PR required for dim-light, peripheral and high-sensibility vision, while cones are involved in normal or high light, acute, central and color vision.
PR present a hazardous environment, resulting from a high exposure to light and a strong presence of free radicals due to their high oxygen consumption, but because these cells are post-mitotic neurons, they have developed a unique mechanism, to avoid damage. Indeed, PR and more precisely OS are continuously regenerated. Around 10% of OS disks are phagocytosed in a circadian manner every day by the RPE, and immediately replaced by new components produced at the base of the OS40, which necessitates the synthesis of up to 10^7 new molecules of rhodopsin/OS/day.
Rods represent the majority of retinal photoreceptors. They are absent from only two distinct regions: the fovea where cones are the only photoreceptors, and the optic disc, the blind point of the eye, which corresponds to the head of the optic nerve.
Retinitis pigmentosa (RP) make up a heterogeneous group of inherited retinal degenerative disorders, firstly characterized by a loss of scotopic vision associated to a mid peripheral visual field failure and usually followed by a central vision loss, leading to blindness. A particularity of this disease is its very important heterogeneity in terms of severity, evolution speed, age of onset and gene mutation, which makes of it a complex disease to study. Although considered as a rare disease due to the small number of patients for each gene, the overall prevalence of RP is around 1 in 4000, making it one of the most common causes of visual impairment in all age groups, and the most prevalent among the working population in developed countries41.
Remodeling of the retina and clinical manifestations
The retinal degeneration observed in RP is primarily due to the death of rod PR, which is then followed by the death of cone PR. The first observation is the shortening of the rod OS, often associated to connecting cilium dysfunction and sprouting of rod neurites, which appears before the apoptotic death of the cell. Cones then follow the same fate, but this is not the only cell lineage to be affected; the subretinal space disappears, due to the migration of RPE cells toward the inner side of the retina, the choriocapillaris (the vascular network located under the retina and responsible for providing oxygen and nutrients to the RPE/PR layers) degenerates and Müller cells enter in an active gliosis phase42. The culmination of all these phenomena lead to the outer retinal degeneration that causes the vision loss.
Patients with RP firstly present a defective dark adaptation, accompanied by mid-peripheral vision loss. These two symptoms are due to the death of rods and constitute the firsts manifestations of the disease that are often hardly noticeable by the affected individuals. The course of the disease leads more or less rapidly to constriction of the visual field, which occurs when most of rods have degenerated. Central visual acuity is often preserved until later stages of the disease, even if cones are also partly damaged.
RP is mainly diagnosed by observation of the fundus appearance, and functional testing of the retina. Some changes are often observed in the fundus, but are not specific to RP, like arteriolar narrowing, retinal vessel attenuation, waxy pallor of the optic nerve, posterior subcapsular cataracts, dust-like particles in the vitreous, white dots deep in the retina…43 The most famous and typical modification observed in RP, giving its name to the disease, is the loss of pigment from the RPE, which form intraretinal clumps of melanin that appear like black spots on the retina (Figure 6). Optical Coherence Tomography (OCT) can also be used to measure retinal thickness and thus follow the degeneration course. It consists in an interferometric technique, typically employing near-infrared light, to penetrate deeply into tissues. It captures micrometer-resolution images, which allow identification of each layer of the retina with RP patients presenting a reduction in thickness of ONL and PR segments layers.
In addition to these purely descriptive aspects, functional criteria also participate to RP diagnosis. Testing of the visual field is used to determine presence of scotomas (blind spots) corresponding to regions where PR have degenerated. Electroretinograms (ERG) are also very informative regarding the functional state of PR. After dark-adaptation, electrical potentials in response to light are recorded by electrodes placed in contact with the cornea. Dim-light stimulation allows measurement of the rod efficiency, and cone function is measured in normal light conditions. Electrical signals are representative of the general state of the retina (focal ERG) or a particular region of this one (multifocal ERG). Patients with RP present rod and cone delayed responses with reduced amplitudes (Figure 7).
RP can be syndromic44 (affecting other neurosensory systems or even systemic, affecting multiple tissues), or non-syndromic (affecting only vision). About 20-30% of RP patients present syndromic RP, listed in more than 30 syndromes, which associate non-ocular disorders, including:
∀ Usher syndrome, which represents 10-20% of RP, and is characterized by hearing loss45,
∀ Bardet-Bield syndrome46,47, in which RP is variably associated with obesity, cognitive impairment, polydactyly, hypogenitalism and retinal disease, accounts for 5-6% of RP cases.
∀ And many other rare syndromes including:
– Refsum’s disease48, in which RP is systematically associated with anosmia and often other variable symptoms,
– Bassen-Kornzweig syndrome, caused by abetalipoproteinaemia49, inducing among other symptoms: balance and coordination difficulties, developmental delay, muscle weakness…
– α-tocopherol transport protein deficiency50, causing ataxia with vitamin E deficiency.
In non-syndromic forms, RP is genetically heterogeneous, including autosomal dominant, RP=retinitis pigmentosa. a=a wave. b=b wave. Vertical dotted lines (left and centre columns) and vertical shock artifacts (right column) represent stimuli. Arrows indicate response times (called implicit times). Extracted from Hartong et al., The Lancet, 2006.
autosomal recessive, X-linked, sporadic and very rare digenic forms. Autosomal dominant RP (adRP) is the most common form of the disease with a prevalence of around 20% as documented in the literature. There is great heterogeneity regarding the degeneration speed and impact on the retina depending on the gene and the mutation implicated, knowing that 25 genes have been associated to adRP43 to date (RetNet, https://sph.uth.edu/retnet/) (Figure 8) with environmental factors also affecting outcomes. Genes already characterized in adRP mainly encode for proteins involved in phototransduction, visual cycle, PR structure or gene expression, but more surprisingly, there is also an important group of genes that encode for proteins implicated in splicing function (PrpF group) and there is no real evidence to date explaining precisely why a mutation on a ubiquitous protein, induces damages only in the eye. Moreover, the involved gene remains unknown in more than 40% of cases. In 1989, the rhodopsin gene (RHO)2 was for the first time found to be linked to adRP by MacWilliam and co-workers51. To date, RHO has been the gene most frequently implicated in adRP with a proportion of 30 to 40% of total adRP occurrences. More than one hundred point mutations of RHO directly associated to adRP have been described, each one being sufficient to develop the disease.
Rhodopsin synthesis, function and visual cycle
While there are three different opsins in cone cells, the RHO gene encodes the only opsin of rod cells. It is firstly synthesized and post-translationally modified by N-Glycosylation into the rough endoplasmic reticulum. After its transport to the Golgi apparatus, proteins are assembled into vesicles, to be exported from the IS through the connecting cilium to attain the OS, where rhodopsin proteins (RHO) are directly integrated into newly synthesized disc membranes. RHO is a G protein-coupled receptor, with 7 trans-membrane domains, which must be associated to the 11-cis-retinal chromophore, a derivative of vitamin A, to be functional. This molecule is covalently bound via a protonated Schiff base to the polypeptide chain of RHO, creating the photopigment of the rods. When the light enters into the retina, photons are absorbed by the 11-cis-retinal, driving to its isomerization to all-trans-retinal isomer. This reaction is the first step of the activation of phototransduction cascade (Figure 9), which transforms the light stimulus caught by the retina into an electrochemical signal that will be transmitted toward the brain. Firstly, some modifications on RHO structure appear, leading to core opening, which permit identification of metarhodopsin I then II52. As an activated G-protein coupled receptor, metarhodopsin II then activates transducin, a trimeric G protein, composed of α, β and γ subunits. The activation of transducin ind uces the exchange of a guanosine diphosphate for a guanosine triphosphate (GTP) and the subsequent dissociation of transducin α-subunit-GTP from the rest of the complex. Transducin α-subunit-GTP in turn allows the activation of the phosphodiesterase. This last step transforms the cyclic guanosine monophosphate (cGMP) present in rods into GMP, leading to closure of cGMP-gated Na+/Ca2+ membrane channels. Finally, this disruption in exchanges across the membrane promotes its hyperpolarization stopping glutamate release. Indeed, in a basal state, i.e. without light stimulus, glutamate is released and PR are depolarized; while light stimuli induce an hyperpolarization. Once activated, RHO also becomes the target of the kinase arrestin, which initiates and completes its inhibition. After a period of time, this complex dissociates, and RHO is dephosphorylated by a phosphatase. RHO is thus available again to bind 11-cis-retinal and to react to light-exposure after about 400s53. On its side, all-trans-retinal is regenerated to be available again for binding to opsin by a series of reactions taking place both in the PR and the RPE cells, and which bear the name of « visual or retinoid cycle »54.
Table of contents :
I. RETINITIS PIGMENTOSA INDUCED BY RHODOPSIN MUTATION
I.1. EYE AND VISION
I.1.b. The retina
I.1.c. Human photoreceptors
I.2. RETINITIS PIGMENTOSA
I.2.a. Remodeling of the retina and clinical manifestations
I.2.b. Genetic involvement
I.3.a. Rhodopsin synthesis, function and visual cycle
I.3.b. Structure of rhodopsin
I.3.c. Rhodopsin mutations and consequences
I.4. TREATMENTS OF RP
I.4.a. General current treatments of RP
I.4.b. Treatments of RP in the particular case of RHO mutation
II. CURRENT STRATEGIES TO PREVENT MUTANT RHODOPSIN EXPRESSION
II.1. RHODOPSIN DNA SEQUENCE CORRECTION
II.1.a. DNA repair by Homologous Recombination (HR)
II.1.b. HR-induced endonucleases
II.2. SILENCING OF MUTANT GENE EXPRESSION COUPLED TO EXPRESSION OF A NORMAL COPY
II.2.a. Zinc Finger – Artificial Transcription Factors (ZF-ATF)
II.2.b. RNA interference
III. TRANS-SPLICING OF RHODOPSIN PRE-MRNA
III.1. CIS-SPLICING: THE PROTOTYPICAL SPLICE REACTION
III.1.a. Splicing is a phase of pre-mRNA maturation
III.1.b. Steps of the splicing mechanism
III.1.c. Regulation of cis-splicing
III.2. TRANS-SPLICING BETWEEN TWO RNA MOLECULES
III.2.a. Description of the mechanism
III.2.b. Previous studies led with trans-splicing approaches
III.2.c. Strategies of PTM Design
III.2.d. Advantages and drawbacks
IV. VIRAL VECTORS FOR GENE THERAPY OF OCULAR DISEASES
IV.1. ADVANTAGES OF THE EYE AS TARGET OF GENE THERAPY WITH VIRAL VECTOR
IV.2. CHOICE OF THE VIRAL VECTOR FOR OCULAR DISEASES
IV.2.a. Classical viral vectors used in gene therapies
IV.2.b. Previous ocular gene therapies with rAAV
V. CELLULAR AND ANIMAL MODELS OF RP LINKED TO RHODOPSIN MUTATIONS.
V.1. CELLULAR MODEL OF RHODOPSIN EXPRESSION
V.1.a. Primary cultures of photoreceptors
V.1.b. Cell lines developed to study rhodopsin mutations
V.2. ANIMAL MODELS OF RHODOPSIN-INDUCED RETINITIS PIGMENTOSA
V.2.a. Animal models of rhodopsin mutation-induced retinitis pigmentosa and more specifically mouse models
V.2.b. Comparison of mouse and human eyes
I. RESULTS OF IN VITRO ANALYSIS OF PTM EFFICIENCY.
II. IMPROVEMENT AND CHARACTERIZATION OF AN IN VIVO IMAGING TECHNOLOGY TO
MEASURE POTENTIAL EFFECTS OF OUR THERAPEUTIC STRATEGY IN A HUMANIZED MOUSE MODEL.
III. COMPLEMENTARY ONGOING RESULTS.
III.1. DEVELOPMENT OF NEW PTM IN VITRO AND STRATEGIES TO OVERCOME PTM DRAWBACKS.
III.1.a. A new PTM that repairs the fifth exon.
III.1.b. Production of a truncated protein in vitro after expression of the PTM alone.
III.1.c. Strategies to prevent production of truncated protein.
III.1.d. Analysis of PTM translation alone in vivo.
III.2. STUDY OF THE THERAPEUTIC EFFECTS OF TRANS-SPLICING IN A HUMANIZED MOUSE MODEL
III.2.a. The humanized Rho+/- P347S RHO+ mouse model.
III.2.b. Choice of parameters that regulate PTM expression in vivo.
III.2.c. Effect of AAV2/8 bRho-PTM20 injection in Rho+/- P347S RHO+ mice.
DISCUSSION AND PERSPECTIVES
I. THE PTM SEQUENCE: THE KEY TO EFFICIENT SMART TECHNOLOGY.
II. HOW TO ACHIEVE MORE TRANS-SPLICING THAN CIS-SPLICING?
III. THE MAIN DRAWBACKS OF PTM.
IV. FOLLOWING-UP POTENTIAL BENEFICIAL EFFECTS IN A HUMANIZED MOUSE MODEL
V. ENVISAGING A THERAPEUTIC APPLICATION IN HUMANS.
VI. THE FUTURE OF THERAPEUTIC STRATEGIES TO PREVENT MUTANT RHODOPSIN EXPRESSION.
VII. CONSIDERING GENE THERAPIES FOR RETINAL DISEASES OVER THE NEXT FEW DECADES.