Gene therapy using recombinant adeno‐associated virus (rAAV) vectors to treat blinding retinal dystrophies has become clinical reality. Therapeutically impactful targeting of photoreceptors still ...relies on subretinal vector delivery, which detaches the retina and harbours substantial risks of collateral damage, often without achieving widespread photoreceptor transduction. Herein, we report the development of novel engineered rAAV vectors that enable efficient targeting of photoreceptors via less invasive intravitreal administration. A unique in vivo selection procedure was performed, where an AAV2‐based peptide‐display library was intravenously administered in mice, followed by isolation of vector DNA from target cells after only 24 h. This stringent selection yielded novel vectors, termed AAV2.GL and AAV2.NN, which mediate widespread and high‐level retinal transduction after intravitreal injection in mice, dogs and non‐human primates. Importantly, both vectors efficiently transduce photoreceptors in human retinal explant cultures. As proof‐of‐concept, intravitreal Cnga3 delivery using AAV2.GL lead to cone‐specific expression of Cnga3 protein and rescued photopic cone responses in the Cnga3−/− mouse model of achromatopsia. These novel rAAV vectors expand the clinical applicability of gene therapy for blinding human retinal dystrophies.
Synopsis
Ocular gene therapy aims to improve or preserve vision in patients with inherited blinding disorders. The current technology still relies on subretinal administration of therapeutic vectors, which harbours risks of collateral damage and only treats a small portion of the affected retina. This study presents two novel engineered viral vectors capable of widespread targeting of retinal cells through a less invasive delivery route.
Novel vectors AAV2.GL and AAV2.NN achieve widespread photoreceptor transduction in mouse, dog and non‐human primate after single intravitreal delivery.
AAV2.GL and AAV2.NN have cone and rod photoreceptor tropism.
AAV2.NN outperforms existing vectors in targeting rod photoreceptors in mice.
Gene supplementation using AAV2.GL rescues cone function and CNGA3 protein expression in the Cnga3−/− mouse model of achromatopsia.
Ocular gene therapy aims to improve or preserve vision in patients with inherited blinding disorders. The current technology still relies on subretinal administration of therapeutic vectors, which harbours risks of collateral damage and only treats a small portion of the affected retina. This study presents two novel engineered viral vectors capable of widespread targeting of retinal cells through a less invasive delivery route.
Cyclic nucleotide‐gated channel β1 (CNGB1) encodes the 240‐kDa β subunit of the rod photoreceptor cyclic nucleotide‐gated ion channel. Disease‐causing sequence variants in CNGB1 lead to autosomal ...recessive rod‐cone dystrophy/retinitis pigmentosa (RP). We herein present a comprehensive review and analysis of all previously reported CNGB1 sequence variants, and add 22 novel variants, thereby enlarging the spectrum to 84 variants in total, including 24 missense variants (two of which may also affect splicing), 21 nonsense, 19 splicing defects (7 at noncanonical positions), 10 small deletions, 1 small insertion, 1 small insertion–deletion, 7 small duplications, and 1 gross deletion. According to the American College of Medical Genetics and Genomics classification criteria, 59 variants were considered pathogenic or likely pathogenic and 25 were variants of uncertain significance. In addition, we provide further phenotypic data from 34 CNGB1‐related RP cases, which, overall, are in line with previous findings suggesting that this form of RP has long‐term retention of useful central vision despite the early onset of night blindness, which is valuable for patient counseling, but also has implications for it being considered a priority target for gene therapy trials.
CNG channel-related retinitis pigmentosa Gerhardt, Maximilian J.; Petersen-Jones, Simon M.; Michalakis, Stylianos
Vision research (Oxford),
07/2023, Letnik:
208
Journal Article
Recenzirano
Odprti dostop
The genes CNGA1 and CNGB1 encode the alpha and beta subunits of the rod CNG channel, a ligand-gated cation channel whose activity is controlled by cyclic guanosine monophosphate (cGMP). Autosomal ...inherited mutations in either of the genes lead to a progressive rod-cone retinopathy known as retinitis pigmentosa (RP). The rod CNG channel is expressed in the plasma membrane of the outer segment and functions as a molecular switch that converts light-mediated changes in cGMP into a voltage and Ca2+ signal. Here, we will first review the molecular properties and physiological role of the rod CNG channel and then discuss the characteristics of CNG-related RP. Finally, we will summarize recent activities in the field of gene therapy aimed at developing therapies for CNG-related RP.
To investigate whether raised levels of retinal cyclic guanosine monophosphate (cGMP) was reflected in plasma levels in PDE6A-/- dogs.
Retina was collected from 2-month-old wildtype dogs (PDE6A+/+, N ...= 6), heterozygous dogs (PDE6A+/-, N = 4) and affected dogs (PDE6A-/-, N = 3) and plasma was collected from 2-month-old wildtype dogs (PDE6A+/+, N = 5), heterozygous dogs (PDE6A+/-, N = 5) and affected dogs (PDE6A-/-, N = 5). Retina and plasma samples were measured by ELISA.
cGMP levels in retinal samples of PDE6A-/- dogs at 2 months of age were significantly elevated. There was no significant difference in plasma cGMP levels between wildtype and PDE6A-/- or PDE6A+/- puppies. However, the plasma cGMP levels of the PDE6A-/- puppies were significantly lower than that of PDE6A+/- puppies.
cGMP levels in the plasma from PDE6A-/- was not elevated when compared to control dogs. At the 2-month timepoint, cGMP plasma levels would not be a useful biomarker for disease.
The On-Off, or long flash, full field electroretinogram (ERG) separates retinal responses to flash onset and offset. Depending on degree of dark-adaptation and stimulus strength the On and Off ERG ...can be shaped by rod and cone photoreceptors and postreceptoral cells, including ON and OFF bipolar cells. Interspecies differences have been shown, with predominantly positive Off-response in humans and other primates and a negative Off-response in rodents and dogs. However, the rod signaling pathways that contribute to these differential responses have not been characterized. In this study, we designed a long flash protocol in the dog that varied in background luminance and stimulus strength allowing for some rod components to be present to better characterize how rod pathways vary from scotopic to mesopic conditions.
With low background light the rod a-wave remains while the b-wave is significantly reduced resulting in a predominantly negative waveform in mesopic conditions. Through modeling and subtraction of the rod-driven response, we show that rod bipolar cells saturate with dimmer backgrounds than rod photoreceptors, resulting in rod hyperpolarization contributing to a large underlying negativity with mesopic backgrounds.
Reduction in rod bipolar cell responses in mesopic conditions prior to suppression of rod photoreceptor responses may reflect the changes in signaling pathway of rod-driven responses needed to extend the range of lighting conditions over which the retina functions.
Electroretinography (ERG) is a commonly used technique to study retinal function in both clinical and research ophthalmology. ERG responses can be divided into component waveforms, analysis of which ...can provide insight into the health and function of different types and populations of retinal cells. In dogs, ERG has been used in the characterization of normal retinal function, as well as the diagnosis of retinal diseases and measuring effects of treatment. While many components of the recorded waveform are similar across species, dogs have several notable features that should be differentiated from the responses in humans and other animals. Additionally, modifications of standard protocols, such as changing flash frequency and stimulus color, and mathematical models of ERG waveforms have been used in studies of human retinal function but have been infrequently applied to visual electrophysiology in dogs. This review provides an overview of the origins and applications of ERG in addition to potential avenues for further characterization of responses in the dog.
Subretinal injections are not commonly performed during clinical treatment of animals but are frequently used in laboratory animal models to assess therapeutic efficacy and safety of gene and cell ...therapy products. Veterinary ophthalmologists are often employed to perform the injections in the laboratory animal setting, due to knowledge of comparative ocular anatomy between species and familiarity with operating on non-human eyes. Understanding the different approaches used for subretinal injection in each species and potential complications that may be encountered is vital to achieving successful and reproducible results. This manuscript provides a summary of different approaches to subretinal injections in the most common animal model species, along with information from published literature and experience of the authors to educate novice or experienced surgeons tasked with performing these injections for the first time.
CRX is a transcription factor essential for normal photoreceptor development and survival. The CRXRdy cat has a naturally occurring truncating mutation in CRX and is a large animal model for dominant ...Leber congenital amaurosis. This study investigated retinal remodeling that occurs as photoreceptors degenerate. CRXRdy/+ cats from 6 weeks to 10 years of age were investigated. In vivo structural changes of retinas were analyzed by fundus examination, confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Histologic analyses included immunohistochemistry for computational molecular phenotyping with macromolecules and small molecules. Affected cats had a cone-led photoreceptor degeneration starting in the area centralis. Initially there was preservation of inner retinal cells such as bipolar, amacrine and horizontal cells but with time migration of the deafferented neurons occurred. Early in the process of degeneration glial activation occurs ultimately resulting in formation of a glial seal. With progression the macula-equivalent area centralis developed severe atrophy including loss of retinal pigmentary epithelium. Microneuroma formation occured in advanced stages as more marked retinal remodeling occurred. This study indicates that retinal degeneration in the CrxRdy/+ cat retina follows the progressive, phased revision of retina that have been previously described for retinal remodeling. These findings suggest that therapy dependent on targeting inner retinal cells may be useful in young adults with preserved inner retinas prior to advanced stages of retinal remodeling and neuronal cell loss.
•CRXRdy/+ cat LCA model follows established stages for retinal remodeling•Area centralis (macula-equivalent) degenerates prior to other retinal regions•Inner retina is initially well preserved•Müller cell activation occurs early and contributes to glial scar formation•Marked remodeling has implications for therapeutic approaches.
Companion animals, namely dogs, cats, and horses, can be affected with many forms of hereditary retinal disease. The number of such diseases characterized in the last decade has increased ...substantially, and nomenclature is nonstandardized, heterogenous, and confusing. We provide in this viewpoint article consensus guidelines for naming of companion animal hereditary retinal diseases, either prospectively or retrospectively. These consensus guidelines have been developed with the purpose of standardizing nomenclature. We provide examples for the iterative nomenclature process and a comprehensive File S1 on proposed renaming of previously described diseases.
Retinal organoid technology enables generation of an inexhaustible supply of three-dimensional retinal tissue from human pluripotent stem cells (hPSCs) for regenerative medicine applications. The ...high similarity of organoid-derived retinal tissue and transplantable human fetal retina provides an opportunity for evaluating and modeling retinal tissue replacement strategies in relevant animal models in the effort to develop a functional retinal patch to restore vision in patients with profound blindness caused by retinal degeneration. Because of the complexity of this very promising approach requiring specialized stem cell and grafting techniques, the tasks of retinal tissue derivation and transplantation are frequently split between geographically distant teams. Delivery of delicate and perishable neural tissue such as retina to the surgical sites requires a reliable shipping protocol and also controlled temperature conditions with damage-reporting mechanisms in place to prevent transplantation of tissue damaged in transit into expensive animal models. We have developed a robust overnight tissue shipping protocol providing reliable temperature control, live monitoring of the shipment conditions and physical location of the package, and damage reporting at the time of delivery. This allows for shipping of viable (transplantation-competent) hPSC-derived retinal tissue over large distances, thus enabling stem cell and surgical teams from different parts of the country to work together and maximize successful engraftment of organoid-derived retinal tissue. Although this protocol was developed for preclinical in vivo studies in animal models, it is potentially translatable for clinical transplantation in the future and will contribute to developing clinical protocols for restoring vision in patients with retinal degeneration.
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