Development of an optimized AAV2/5 gene therapy vector for Leber congenital amaurosis owing to defects in RPE65

Abstract Leber congenital amaurosis is a group of inherited retinal dystrophies that cause severe sight impairment in childhood; RPE65-deficiency causes impaired rod photoreceptor function from birth and progressive impairment of cone photoreceptor function associated with retinal degeneration. In animal models of RPE65 deficiency, subretinal injection of recombinant adeno-associated virus (AAV) 2/2 vectors carrying RPE65 cDNA improves rod photoreceptor function, and intervention at an early stage of disease provides sustained benefit by protecting cone photoreceptors against retinal degeneration. In affected humans, administration of these vectors has resulted to date in relatively modest improvements in photoreceptor function, even when retinal degeneration is comparatively mild, and the duration of benefit is limited by progressive retinal degeneration. We conclude that the demand for RPE65 in humans is not fully met by current vectors, and predict that a more powerful vector will provide more durable benefit. With this aim we have modified the original AAV2/2 vector to generate AAV2/5-OPTIRPE65. The new configuration consists of an AAV vector serotype 5 carrying an optimized hRPE65 promoter and a codon-optimized hRPE65 gene. In mice, AAV2/5-OPTIRPE65 is at least 300-fold more potent than our original AAV2/2 vector.

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Carbon Dot Nanoparticles: Exploring the Potential Use for Gene Delivery in Ophthalmic Diseases

Nanomaterials ◽

10.3390/nano11040935 ◽

2021 ◽

Vol 11 (4) ◽

pp. 935

Author(s):

Manas R. Biswal ◽

Sofia Bhatia

Keyword(s):

Gene Delivery ◽

Therapeutic Option ◽

Retinal Dystrophy ◽

Retinal Cells ◽

Adeno Associated Virus ◽

Inherited Retinal Dystrophies ◽

Retinal Dystrophies ◽

Efficient Gene ◽

New Type ◽

Viral Nanoparticles

Ocular gene therapy offers significant potential for preventing retinal dystrophy in patients with inherited retinal dystrophies (IRD). Adeno-associated virus (AAV) based gene transfer is the most common and successful gene delivery approach to the eye. These days, many studies are using non-viral nanoparticles (NPs) as an alternative therapeutic option because of their unique properties and biocompatibility. Here, we discuss the potential of carbon dots (CDs), a new type of nanocarrier for gene delivery to the retinal cells. The unique physicochemical properties of CDs (such as optical, electronic, and catalytic) make them suitable for biosensing, imaging, drug, and gene delivery applications. Efficient gene delivery to the retinal cells using CDs depends on various factors, such as photoluminescence, quantum yield, biocompatibility, size, and shape. In this review, we focused on different approaches used to synthesize CDs, classify CDs, various pathways for the intake of gene-loaded carbon nanoparticles inside the cell, and multiple studies that worked on transferring nucleic acid in the eye using CDs.

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Nutraceutical Supplementation Ameliorates Visual Function, Retinal Degeneration, and Redox Status in rd10 Mice

Antioxidants ◽

10.3390/antiox10071033 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1033

Author(s):

Lorena Olivares-González ◽

Sheyla Velasco ◽

Isabel Campillo ◽

David Salom ◽

Emilio González-García ◽

...

Keyword(s):

Oxidative Stress ◽

Retinal Degeneration ◽

Antioxidant Properties ◽

Photoreceptor Cells ◽

Redox Status ◽

Inherited Retinal Dystrophies ◽

Stress Markers ◽

Retinal Dystrophies ◽

Progressive Degeneration ◽

Light Stimuli

Background: Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies characterized by progressive degeneration of photoreceptor cells. Ocular redox status is altered in RP suggesting oxidative stress could contribute to their progression. In this study, we investigated the effect of a mixture of nutraceuticals with antioxidant properties (NUT) on retinal degeneration in rd10 mice, a model of RP. Methods: NUT was orally administered to rd10 mice from postnatal day (PD) 9 to PD18. At PD18 retinal function and morphology were examined by electroretinography (ERG) and histology including TUNEL assay, immunolabeling of microglia, Müller cells, and poly ADP ribose polymers. Retinal redox status was determined by measuring the activity of antioxidant enzymes and some oxidative stress markers. Gene expression of the cytokines IL-6, TNFα, and IL-1β was assessed by real-time PCR. Results: NUT treatment delayed the loss of photoreceptors in rd10 mice partially preserving their electrical responses to light stimuli. Moreover, it ameliorated redox status and reduced inflammation including microglia activation, upregulation of cytokines, reactive gliosis, and PARP overactivation. Conclusions: NUT ameliorated retinal functionality and morphology at early stages of RP in rd10 mice. This formulation could be useful as a neuroprotective approach for patients with RP in the future.

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Phase I Trial of Leber Congenital Amaurosis due to RPE65 Mutations by Ocular Subretinal Injection of Adeno-Associated Virus Gene Vector: Short-Term Results

Human Gene Therapy ◽

10.1089/hgt.2008.107 ◽

2008 ◽

Vol 0 (ja) ◽

pp. 081015093227032 ◽

Cited By ~ 4

Author(s):

William Hauswirth ◽

Tomas S Aleman ◽

Shalesh Kaushal ◽

Artur V Cideciyan ◽

Sharon B Schwartz ◽

...

Keyword(s):

Phase I ◽

Phase I Trial ◽

Gene Vector ◽

Short Term ◽

Adeno Associated Virus ◽

Leber Congenital Amaurosis ◽

Virus Gene ◽

Subretinal Injection

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CRISPR-Cas9–mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis

Science Advances ◽

10.1126/sciadv.aax1210 ◽

2019 ◽

Vol 5 (10) ◽

pp. eaax1210 ◽

Cited By ~ 8

Author(s):

Dong Hyun Jo ◽

Dong Woo Song ◽

Chang Sik Cho ◽

Un Gi Kim ◽

Kyu Jun Lee ◽

...

Keyword(s):

Nonsense Mutation ◽

Stop Codon ◽

Retinal Pigment ◽

Childhood Onset ◽

Adeno Associated Virus ◽

Leber Congenital Amaurosis ◽

Homology Directed Repair ◽

Disease Phenotypes ◽

Recessive Mutations ◽

Subretinal Injection

Leber congenital amaurosis (LCA), one of the leading causes of childhood-onset blindness, is caused by autosomal recessive mutations in several genes including RPE65. In this study, we performed CRISPR-Cas9–mediated therapeutic correction of a disease-associated nonsense mutation in Rpe65 in rd12 mice, a model of human LCA. Subretinal injection of adeno-associated virus carrying CRISPR-Cas9 and donor DNA resulted in >1% homology-directed repair and ~1.6% deletion of the pathogenic stop codon in Rpe65 in retinal pigment epithelial tissues of rd12 mice. The a- and b-waves of electroretinograms were recovered to levels up to 21.2 ± 4.1% and 39.8 ± 3.2% of their wild-type mice counterparts upon bright stimuli after dark adaptation 7 months after injection. There was no definite evidence of histologic perturbation or tumorigenesis during 7 months of observation. Collectively, we present the first therapeutic correction of an Rpe65 nonsense mutation using CRISPR-Cas9, providing new insight for developing therapeutics for LCA.

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On the Wrong Track: Alterations of Ciliary Transport in Inherited Retinal Dystrophies

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.623734 ◽

2021 ◽

Vol 9 ◽

Author(s):

Laura Sánchez-Bellver ◽

Vasileios Toulis ◽

Gemma Marfany

Keyword(s):

Retinal Degeneration ◽

Outer Segment ◽

Molecular Mechanisms ◽

Photoreceptor Cells ◽

Cellular Functions ◽

Inherited Disorders ◽

Inherited Retinal Dystrophies ◽

Retinal Dystrophies ◽

Elevated Protein ◽

And Function

Ciliopathies are a group of heterogeneous inherited disorders associated with dysfunction of the cilium, a ubiquitous microtubule-based organelle involved in a broad range of cellular functions. Most ciliopathies are syndromic, since several organs whose cells produce a cilium, such as the retina, cochlea or kidney, are affected by mutations in ciliary-related genes. In the retina, photoreceptor cells present a highly specialized neurosensory cilium, the outer segment, stacked with membranous disks where photoreception and phototransduction occurs. The daily renewal of the more distal disks is a unique characteristic of photoreceptor outer segments, resulting in an elevated protein demand. All components necessary for outer segment formation, maintenance and function have to be transported from the photoreceptor inner segment, where synthesis occurs, to the cilium. Therefore, efficient transport of selected proteins is critical for photoreceptor ciliogenesis and function, and any alteration in either cargo delivery to the cilium or intraciliary trafficking compromises photoreceptor survival and leads to retinal degeneration. To date, mutations in more than 100 ciliary genes have been associated with retinal dystrophies, accounting for almost 25% of these inherited rare diseases. Interestingly, not all mutations in ciliary genes that cause retinal degeneration are also involved in pleiotropic pathologies in other ciliated organs. Depending on the mutation, the same gene can cause syndromic or non-syndromic retinopathies, thus emphasizing the highly refined specialization of the photoreceptor neurosensory cilia, and raising the possibility of photoreceptor-specific molecular mechanisms underlying common ciliary functions such as ciliary transport. In this review, we will focus on ciliary transport in photoreceptor cells and discuss the molecular complexity underpinning retinal ciliopathies, with a special emphasis on ciliary genes that, when mutated, cause either syndromic or non-syndromic retinal ciliopathies.

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Crumbs homologue 1 in polarity and blindness

Biochemical Society Transactions ◽

10.1042/bst0320828 ◽

2004 ◽

Vol 32 (5) ◽

pp. 828-830 ◽

Cited By ~ 10

Author(s):

J. Meuleman ◽

S.A. van de Pavert ◽

J. Wijnholds

Keyword(s):

Retinitis Pigmentosa ◽

Retinal Degeneration ◽

Cell Polarity ◽

Current Knowledge ◽

Leber Congenital Amaurosis ◽

Retinal Dystrophies

Several retinal dystrophies, including retinitis pigmentosa type 12 and Leber congenital amaurosis, are caused by a large variety of mutations in the CRB1 (Crumbs homologue 1) gene. This discovery led to an increased focus on the function of CRB1 and the Drosophila homologue Crumbs. In the present study, we review the current knowledge on Crumbs and its vertebrate homologues, their function in cell polarity and their pathogenicity in retinal degeneration.

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Toward the Treatment of Inherited Diseases of the Retina Using CRISPR-Based Gene Editing

Frontiers in Medicine ◽

10.3389/fmed.2021.698521 ◽

2021 ◽

Vol 8 ◽

Author(s):

Jennifer Hernández-Juárez ◽

Genaro Rodríguez-Uribe ◽

Shyamanga Borooah

Keyword(s):

Gene Editing ◽

Vision Loss ◽

Therapeutic Modality ◽

Leber Congenital Amaurosis ◽

Inherited Diseases ◽

Inherited Retinal Dystrophies ◽

Retinal Dystrophies ◽

Severe Vision Loss ◽

Targeted Gene Editing

Inherited retinal dystrophies [IRDs] are a common cause of severe vision loss resulting from pathogenic genetic variants. The eye is an attractive target organ for testing clinical translational approaches in inherited diseases. This has been demonstrated by the approval of the first gene supplementation therapy to treat an autosomal recessive IRD, RPE65-linked Leber congenital amaurosis (type 2), 4 years ago. However, not all diseases are amenable for treatment using gene supplementation therapy, highlighting the need for alternative strategies to overcome the limitations of this supplementation therapeutic modality. Gene editing has become of increasing interest with the discovery of the CRISPR-Cas9 platform. CRISPR-Cas9 offers several advantages over previous gene editing technologies as it facilitates targeted gene editing in an efficient, specific, and modifiable manner. Progress with CRISPR-Cas9 research now means that gene editing is a feasible strategy for the treatment of IRDs. This review will focus on the background of CRISPR-Cas9 and will stress the differences between gene editing using CRISPR-Cas9 and traditional gene supplementation therapy. Additionally, we will review research that has led to the first CRISPR-Cas9 trial for the treatment of CEP290-linked Leber congenital amaurosis (type 10), as well as outline future directions for CRISPR-Cas9 technology in the treatment of IRDs.

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An inherited night blindness in Wiltshire sheep

Veterinary Pathology ◽

10.1177/03009858211067461 ◽

2022 ◽

pp. 030098582110674

Author(s):

Hayley Hunt ◽

Keren E. Dittmer ◽

Dorian J. Garrick ◽

Robert A. Fairley ◽

Stephen J. Heap ◽

...

Keyword(s):

Retinal Degeneration ◽

Night Blindness ◽

Autosomal Recessive Inheritance ◽

Homozygosity Mapping ◽

Pedigree Analysis ◽

Rod Photoreceptors ◽

Progressive Retinal Atrophy ◽

Inherited Retinal Dystrophies ◽

Retinal Dystrophies ◽

Retinal Blood Vessels

Twelve cases of adult-onset blindness were identified in a flock of 130 polled Wiltshire sheep in New Zealand over a 3-year period. Affected sheep developed night blindness between 2 and 3 years of age, which progressed to complete blindness by 4 to 5 years of age. Fundic examination findings included progressive tapetal hyperreflectivity and attenuation of retinal blood vessels. Histologically, the retinas had a selective loss of rod photoreceptors with initial preservation of cone photoreceptors. Retinal degeneration was not accompanied by any other ocular or central nervous system abnormalities, and pedigree analysis suggested an inherited basis for the disease. Mating an affected Wiltshire ram to 2 affected Wiltshire ewes resulted in 6 progeny that all developed retinal degeneration by 2 years of age, while mating of the same affected ram to 6 unaffected ewes resulted in 8 unaffected progeny, consistent with autosomal recessive inheritance. Homozygosity mapping of 5 affected Wiltshire sheep and 1 unaffected Wiltshire sheep using an OvineSNP50 Genotyping BeadChip revealed an identical-by-descent region on chromosome 5, but none of the genes within this region were considered plausible candidate genes. Whole-genome sequencing of 2 affected sheep did not reveal any significant mutations in any of the genes associated with retinitis pigmentosa in humans or progressive retinal atrophy in dogs. Inherited progressive retinal degeneration affecting rod photoreceptors has not been previously reported in sheep, but this disease has several similarities to inherited retinal dystrophies in other species.

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Enhancer of Zeste Homolog 2 (EZH2) Contributes to Rod Photoreceptor Death Process in Several Forms of Retinal Degeneration and Its Activity Can Serve as a Biomarker for Therapy Efficacy

International Journal of Molecular Sciences ◽

10.3390/ijms22179331 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9331

Author(s):

Martial Mbefo ◽

Adeline Berger ◽

Karine Schouwey ◽

Xavier Gérard ◽

Corinne Kostic ◽

...

Keyword(s):

Cell Death ◽

Retinal Degeneration ◽

Gene Mutations ◽

Cell Number ◽

Death Process ◽

Epigenetic Mark ◽

Specific Cell ◽

Rod Photoreceptor ◽

Inherited Retinal Dystrophies ◽

Cell Death Process

Inherited retinal dystrophies (IRD) are due to various gene mutations. Each mutated gene instigates a specific cell homeostasis disruption, leading to a modification in gene expression and retinal degeneration. We previously demonstrated that the polycomb-repressive complex-1 (PRC1) markedly contributes to the cell death process. To better understand these mechanisms, we herein study the role of PRC2, specifically EZH2, which often initiates the gene inhibition by PRC1. We observed that the epigenetic mark H3K27me3 generated by EZH2 was progressively and strongly expressed in some individual photoreceptors and that the H3K27me3-positive cell number increased before cell death. H3K27me3 accumulation occurs between early (accumulation of cGMP) and late (CDK4 expression) events of retinal degeneration. EZH2 hyperactivity was observed in four recessive and two dominant mouse models of retinal degeneration, as well as two dog models and one IRD patient. Acute pharmacological EZH2 inhibition by intravitreal injection decreased the appearance of H3K27me3 marks and the number of TUNEL-positive cells revealing that EZH2 contributes to the cell death process. Finally, we observed that the absence of the H3K27me3 mark is a biomarker of gene therapy treatment efficacy in XLRPA2 dog model. PRC2 and PRC1 are therefore important actors in the degenerative process of multiple forms of IRD.

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