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

2019 ◽  
Vol 5 (10) ◽  
pp. eaax1210 ◽  
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.

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

2008 ◽  
Vol 0 (ja) ◽  
pp. 081015093227032 ◽  
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

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

Gene Therapy ◽  
2016 ◽  
Vol 23 (12) ◽  
pp. 857-862 ◽  
Author(s):  
A Georgiadis ◽  
Y Duran ◽  
J Ribeiro ◽  
L Abelleira-Hervas ◽  
S J Robbie ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Early Stage ◽  
Rod Photoreceptor ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Leber Congenital Amaurosis ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
Serotype 5 ◽  
Subretinal Injection

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.

scholarly journals Investigation and Restoration of BEST1 Activity in Patient-derived RPEs with Dominant Mutations

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Changyi Ji ◽  
Yao Li ◽  
Alec Kittredge ◽  
Austin Hopiavuori ◽  
Nancy Ward ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Similar Efficacy ◽  
Hek293 Cells ◽  
Macular Dystrophy ◽  
Loss Of Function ◽  
Adeno Associated Virus ◽  
Recessive Mutations ◽  
Degenerative Disorders

AbstractBEST1 is a Ca2+-activated Cl− channel predominantly expressed in retinal pigment epithelium (RPE), and over 250 genetic mutations in the BEST1 gene have been identified to cause retinal degenerative disorders generally known as bestrophinopathies. As most BEST1 mutations are autosomal dominant, it is of great biomedical interest to determine their disease-causing mechanisms and the therapeutic potential of gene therapy. Here, we characterized six Best vitelliform macular dystrophy (BVMD)-associated BEST1 dominant mutations by documenting the patients’ phenotypes, examining the subcellular localization of endogenous BEST1 and surface Ca2+-dependent Cl− currents in patient-derived RPEs, and analyzing the functional influences of these mutations on BEST1 in HEK293 cells. We found that all six mutations are loss-of-function with different levels and types of deficiencies, and further demonstrated the restoration of Ca2+-dependent Cl− currents in patient-derived RPE cells by WT BEST1 gene supplementation. Importantly, BEST1 dominant and recessive mutations are both rescuable at a similar efficacy by gene augmentation via adeno-associated virus (AAV), providing a proof-of-concept for curing the vast majority of bestrophinopathies.

scholarly journals Therapeutic adenine base editing corrects nonsense mutation and improves visual function in a mouse model of Leber congenital amaurosis

2021 ◽  
Author(s):  
Dong Hyun Jo ◽  
Hyeon-Ki Jang ◽  
Chang Sik Cho ◽  
Jun Hee Han ◽  
Gahee Ryu ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Nonsense Mutation ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Subretinal Space ◽  
Leber Congenital Amaurosis ◽  
Base Editing ◽  
Pathogenic Variants ◽  
Adenine Base

Leber congenital amaurosis (LCA) is an inherited retinal degeneration that causes severe visual dysfunction in children and adolescents. In patients with LCA, pathogenic variants are evident in specific genes, such as RPE65, which are related to the functions of retinal pigment epithelium and photoreceptors. Base editing confers a way to correct pathogenic substitutions without double-stranded breaks in contrast to the original Cas9. In this study, we prepared dual adeno-associated virus vectors containing the split adenine base editors with trans-splicing intein (AAV-ABE) for in vivo adenine base editing in retinal degeneration 12 (rd12) mice, an animal model of LCA, which possess a nonsense mutation of C to T transition in the Rpe65 gene (p.R44X). AAV-ABE induced an A to G transition in retinal pigment epithelial cells of rd12 mice when injected into the subretinal space. The on-target editing was sufficient to recover wild-type mRNA, RPE65 protein, and light-induced electrical responses of retinal tissues. We suggest adenine base editing to correct pathogenic variants in the treatment of LCA.

scholarly journals Treatment of Leber Congenital Amaurosis Due toRPE65Mutations by Ocular Subretinal Injection of Adeno-Associated Virus Gene Vector: Short-Term Results of a Phase I Trial

2008 ◽  
Vol 19 (10) ◽  
pp. 979-990 ◽  
Author(s):  
William W. 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

scholarly journals An Update on Gene Therapy for Inherited Retinal Dystrophy: Experience in Leber Congenital Amaurosis Clinical Trials

2021 ◽  
Vol 22 (9) ◽  
pp. 4534
Author(s):  
Wei Chiu ◽  
Ting-Yi Lin ◽  
Yun-Chia Chang ◽  
Henkie Isahwan-Ahmad Mulyadi Lai ◽  
Shen-Che Lin ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Vision Loss ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Dystrophy ◽  
Clinical Manifestations ◽  
Adeno Associated Virus ◽  
Leber Congenital Amaurosis ◽  
Gene Therapies

Inherited retinal dystrophies (IRDs) are a group of rare eye diseases caused by gene mutations that result in the degradation of cone and rod photoreceptors or the retinal pigment epithelium. Retinal degradation progress is often irreversible, with clinical manifestations including color or night blindness, peripheral visual defects and subsequent vision loss. Thus, gene therapies that restore functional retinal proteins by either replenishing unmutated genes or truncating mutated genes are needed. Coincidentally, the eye’s accessibility and immune-privileged status along with major advances in gene identification and gene delivery systems heralded gene therapies for IRDs. Among these clinical trials, voretigene neparvovec-rzyl (Luxturna), an adeno-associated virus vector-based gene therapy drug, was approved by the FDA for treating patients with confirmed biallelic RPE65 mutation-associated Leber Congenital Amaurosis (LCA) in 2017. This review includes current IRD gene therapy clinical trials and further summarizes preclinical studies and therapeutic strategies for LCA, including adeno-associated virus-based gene augmentation therapy, 11-cis-retinal replacement, RNA-based antisense oligonucleotide therapy and CRISPR-Cas9 gene-editing therapy. Understanding the gene therapy development for LCA may accelerate and predict the potential hurdles of future therapeutics translation. It may also serve as the template for the research and development of treatment for other IRDs.

scholarly journals Translational switching of Cry1 protein expression confers reversible control of circadian behavior in arrhythmic Cry-deficient mice

2018 ◽  
Vol 115 (52) ◽  
pp. E12388-E12397 ◽  
Author(s):  
Elizabeth S. Maywood ◽  
Thomas S. Elliott ◽  
Andrew P. Patton ◽  
Toke P. Krogager ◽  
Johanna E. Chesham ◽  
...  
Keyword(s):  
Stop Codon ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Adeno Associated Virus ◽  
Biologically Relevant ◽  
Negative Feedback Loops ◽  
Translational Readthrough ◽  
Genetic Code Expansion ◽  
And Behavior ◽  
Circadian Behavior

The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals, coordinating daily rhythms of physiology and behavior. Circadian timing pivots around self-sustaining transcriptional–translational negative feedback loops (TTFLs), whereby CLOCK and BMAL1 drive the expression of the negative regulators Period and Cryptochrome (Cry). Global deletion of Cry1 and Cry2 disables the TTFL, resulting in arrhythmicity in downstream behaviors. We used this highly tractable biology to further develop genetic code expansion (GCE) as a translational switch to achieve reversible control of a biologically relevant protein, Cry1, in the SCN. This employed an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair delivered to the SCN by adeno-associated virus (AAV) vectors, allowing incorporation of a noncanonical amino acid (ncAA) into AAV-encoded Cry1 protein carrying an ectopic amber stop codon. Thus, translational readthrough and Cry1 expression were conditional on the supply of ncAA via culture medium or drinking water and were restricted to neurons by synapsin-dependent expression of aminoacyl tRNA-synthetase. Activation of Cry1 translation by ncAA in neurons of arrhythmic Cry-null SCN slices immediately and dose-dependently initiated TTFL circadian rhythms, which dissipated rapidly after ncAA withdrawal. Moreover, genetic activation of the TTFL in SCN neurons rapidly and reversibly initiated circadian behavior in otherwise arrhythmic Cry-null mice, with rhythm amplitude being determined by the number of transduced SCN neurons. Thus, Cry1 does not specify the development of circadian circuitry and competence but is essential for its labile and rapidly reversible activation. This demonstrates reversible control of mammalian behavior using GCE-based translational switching, a method of potentially broad neurobiological interest.

Challenges in non-viral ocular gene transfer

2007 ◽  
Vol 35 (1) ◽  
pp. 47-49 ◽  
Author(s):  
L. Peeters ◽  
N.N. Sanders ◽  
J. Demeester ◽  
S.C. De Smedt
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Viral Gene ◽  
Target Tissue ◽  
Long Distance ◽  
Promising Alternative ◽  
Gene Complexes ◽  
Poly Ethylene ◽  
Review Reports ◽  
Subretinal Injection

Nowadays, there is no effective treatment for many retinal disorders. Knowledge of the genetic basis of many severe ocular diseases may allow for alternative treatments by gene therapy. Non-viral gene complexes, such as lipo- and poly-plexes, can be delivered to the posterior segment, most often the target tissue, by intravitreal or subretinal injection. Since subretinal injections are very invasive, intravitreal injection is a promising alternative route to deliver gene complexes into the eye. However, the drawback of this technique is the relative long distance the complexes have to travel through the vitreous gel before they reach the retina. This mini-review reports on how non-viral gene complexes behave in vitreous. It especially focuses on how the coating of lipoplexes with poly(ethylene glycol) influences their behaviour in vitreous and the transfection of retinal pigment epithelium.

scholarly journals Identification of ATP2C1 mutations in the patients of Hailey-Hailey disease

2020 ◽  
Author(s):  
xiaoli li ◽  
dingwei zhang ◽  
jiahui ding ◽  
li li ◽  
zhenghui wang
Keyword(s):  
Nonsense Mutation ◽  
Polypeptide Chain ◽  
Stop Codon ◽  
Skin Lesions ◽  
Signaling Proteins ◽  
Missense Mutations ◽  
Functional Region ◽  
Bullous Dermatosis ◽  
Normal Human ◽  
Regulatory Effects

Abstract Background: Familial benign chronic pemphigus, also known as Hailey-Hailey disease (HHD), is a clinically rare bullous Dermatosis. However the mechanism has not been clarified. The study aim to detect novel mutations in exons of ATP2C1 gene in HHD patients; to explore the possible mechnism of HHD pathogenesis by examining the expression profile of hSPCA1, miR-203, p63, Notch1 and HKⅡ proteins in the skin lesions of HHD patients. Methods: Genomic DNA was extracted from peripheral blood of HHD patients. All exons of ATP2C1 gene in HHD patients were amplified by PCR and the products were purified and sequenced. All related signaling proteins of interest were stained by using skin lesion tissues from HHD patients and miR-203 levels were also determined. Results: One synonymous mutation c.G2598A (in exon 26), one nonsense mutation c.C635A and two missense mutations c.C1286A (p.A429D) and c. A1931G (p. D644G) were identified. The nonsense mutation changed codon UCG to stop codon UAG, causing a premature polypeptide chain of the functional region A. The two missense mutations were located in the region P (phosphorylation region) and the Mn binding site of hSPCA1. The level of hSPCA1 was significantly decreased in HHD patients compared to the normal human controls, accompanied by an increase of miR-203 level and a decrease of p63 and HKⅡ levels. Conclusion: In our study, we found four mutations in HHD. Meanwhile we found increase of miR-203 level and a decrease of p63 and HKⅡ levels. In addition, Notch1, which was negatively regulated p63, is downregulated. These factors may be involved in the signaling pathways of HHD pathogenesis. Our data showed that both p63 and miR-203 may have significant regulatory effects on Notch1 in the skin.

scholarly journals Identification of ATP2C1 mutations in the patients of Hailey-Hailey disease

2020 ◽  
Author(s):  
xiaoli li ◽  
dingwei zhang ◽  
jiahui ding ◽  
li li ◽  
zhenghui wang
Keyword(s):  
Nonsense Mutation ◽  
Polypeptide Chain ◽  
Stop Codon ◽  
Skin Lesions ◽  
Signaling Proteins ◽  
Missense Mutations ◽  
Functional Region ◽  
Bullous Dermatosis ◽  
Normal Human ◽  
Regulatory Effects

Abstract Background: Familial benign chronic pemphigus, also known as Hailey-Hailey disease (HHD), is a clinically rare bullous Dermatosis. However the mechanism has not been clarified. The study aim to detect novel mutations in exons of ATP2C1 gene in HHD patients; to explore the possible mechnism of HHD pathogenesis by examining the expression profile of hSPCA1, miR-203, p63, Notch1 and HKⅡ proteins in the skin lesions of HHD patients. Methods: Genomic DNA was extracted from peripheral blood of HHD patients. All exons of ATP2C1 gene in HHD patients were amplified by PCR and the products were purified and sequenced. All related signaling proteins of interest were stained by using skin lesion tissues from HHD patients and miR-203 levels were also determined. Results: One synonymous mutation c.G2598A (in exon 26), one nonsense mutation c.C635A and two missense mutations c.C1286A (p.A429D) and c. A1931G (p. D644G) were identified. The nonsense mutation changed codon UCG to stop codon UAG, causing a premature polypeptide chain of the functional region A. The two missense mutations were located in the region P (phosphorylation region) and the Mn binding site of hSPCA1. The level of hSPCA1 was significantly decreased in HHD patients compared to the normal human controls, accompanied by an increase of miR-203 level and a decrease of p63 and HKⅡ levels. Conclusion: In our study, we found four mutations in HHD. Meanwhile we found increase of miR-203 level and a decrease of p63 and HKⅡ levels. In addition, Notch1, which was negatively regulated p63, is downregulated. These factors may be involved in the signaling pathways of HHD pathogenesis. Our data showed that both p63 and miR-203 may have significant regulatory effects on Notch1 in the skin.

Sign in / Sign up

Export Citation Format

Share Document