In vitro Analysis of Ribozyme-mediated Knockdown of an ADRP Associated Rhodopsin Mutation

Chakraborty, Dibyendu; Whalen, Patrick; Lewin, Alfred S.; Naash, Muna I.

doi:10.1007/978-0-387-74904-4_10

Cited by 3 publications

(2 citation statements)

References 34 publications

(42 reference statements)

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“…46 Post-transcriptional protein knockdown has also been attempted through hammerhead and hairpin ribozymes designed to target and cleave P23H, with good specificity in vitro. 47 In addition, a dual-approach to both suppress the mutated gene and supplement a wild type gene is being actively developed. Suppression has been implemented via RNA silencing (e.g.…”

Section: Many Techniques Have Been Explored To Treat Rho-retinopathy ...mentioning

confidence: 99%

Genetic treatment for autosomal dominant inherited retinal dystrophies: approaches, challenges and targeted genotypes

Varela

Georgiadis²,

Michaelides

2022

Br J Ophthalmol

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Inherited retinal diseases (IRDs) have been in the front line of gene therapy development for the last decade, providing a useful platform to test novel therapeutic approaches. More than 40 clinical trials have been completed or are ongoing, tackling autosomal recessive and X-linked conditions, mostly through adeno-associated viral vector delivery of a normal copy of the disease-causing gene. However, only recently has autosomal dominant (ad) disease been targeted, with the commencement of a trial for rhodopsin (RHO)-associated retinitis pigmentosa (RP), implementing antisense oligonucleotide (AON) therapy, with promising preliminary results (NCT04123626).Autosomal dominant RP represents 15%–25% of all RP, with RHO accounting for 20%–30% of these cases. Autosomal dominant macular and cone-rod dystrophies (MD/CORD) correspond to approximately 7.5% of all IRDs, and approximately 35% of all MD/CORD cases, with the main causative gene being BEST1. Autosomal dominant IRDs are not only less frequent than recessive, but also tend to be less severe and have later onset; for example, an individual with RHO-adRP would typically become severely visually impaired at an age 2–3 times older than in X-linked RPGR-RP.Gain-of-function and dominant negative aetiologies are frequently seen in the prevalent adRP genes RHO, RP1 and PRPF31 among others, which would not be effectively addressed by gene supplementation alone and need creative, novel approaches. Zinc fingers, RNA interference, AON, translational read-through therapy, and gene editing by clustered regularly interspaced short palindromic repeats/Cas are some of the strategies that are currently under investigation and will be discussed here.

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Section: Many Techniques Have Been Explored To Treat Rho-retinopathy ...mentioning

confidence: 99%

Genetic treatment for autosomal dominant inherited retinal dystrophies: approaches, challenges and targeted genotypes

Varela

Georgiadis²,

Michaelides

2022

Br J Ophthalmol

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“…This approach has been explored with relative success for rhodopsin gene therapy. Like PRPH2, there are a plethora of different dominant, disease-causing mutant rhodopsin alleles that cause inherited retinal degeneration, and knockdown has been attempted in vitro and in vivo with varying degrees of success using multiple methods, including ribozymes (Chakraborty et al 2008b), siRNA Gorbatyuk et al 2007;O'Reilly et al 2007;Mao et al 2012), and zinc finger nucleases (Mussolino et al 2011). Thus far, shRNA is the most well-developed approach, and recent results suggest that a single AAV vector carrying an shRNA capable of knocking down WT-and mutant rhodopsin, coupled with a WT rhodopsin that is resistant to silencing, results in long-term (up to 9 mo of age) preservation of photoreceptor structure and function in the P23H mouse model of rhodopsin-associated ADRP (Mao et al 2012).…”

Section: Gene Knockdown Therapy In Peripherin-2 Modelsmentioning

confidence: 99%

Gene Therapy for PRPH2-Associated Ocular Disease: Challenges and Prospects

Conley

Naash

2014

Cold Spring Harbor Perspectives in Medicine

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Wild-type opsin does not aggregate with a misfolded opsin mutant

Gragg

Kim

Howell

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Rhodopsin is the light receptor in photoreceptor cells that plays a central role in phototransduction and photoreceptor cell health. Mutations in rhodopsin are the leading cause of autosomal dominant retinitis pigmentosa (adRP), a retinal degenerative disease. A majority of mutations in rhodopsin cause misfolding and aggregation of the apoprotein opsin. The pathogenesis of adRP caused by misfolded opsin is unclear. It has been proposed that physical interactions between wild-type opsin and misfolded opsin mutants may underlie the autosomal dominant phenotype. To test whether or not wild-type opsin can form a complex with misfolded opsin mutants, we examined the interactions between wild-type opsin and opsin with a G188R mutation, a clinically identified mutation causing adRP. Förster resonance energy transfer (FRET) was utilized to monitor the interactions between fluorescently tagged opsins expressed in live cells. The FRET assay employed was able to discriminate between properly folded opsin oligomers and misfolded opsin aggregates. Wild-type opsin predominantly formed oligomers and only a minor population formed aggregates. Conversely, the G188R opsin mutant predominantly formed aggregates. When wild-type opsin and G188R opsin were coexpressed in cells, properly folded wild-type opsin did not aggregate with G188R opsin and was trafficked normally to the plasma membrane. Thus, the autosomal dominant phenotype in adRP caused by misfolded opsin mutants is not predicted to arise from physical interactions between wild-type opsin and misfolded opsin mutants.

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In vitro Analysis of Ribozyme-mediated Knockdown of an ADRP Associated Rhodopsin Mutation

Cited by 3 publications

References 34 publications

Genetic treatment for autosomal dominant inherited retinal dystrophies: approaches, challenges and targeted genotypes

Genetic treatment for autosomal dominant inherited retinal dystrophies: approaches, challenges and targeted genotypes

Gene Therapy for PRPH2-Associated Ocular Disease: Challenges and Prospects

Wild-type opsin does not aggregate with a misfolded opsin mutant

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