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• W3080180587 abstract "Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP) is a hereditary degenerative disorder in which mutations in the gene encoding RHO, the light-sensitive G protein-coupled receptor involved in phototransduction in rods, lead to progressive loss of rods and subsequently cones in the retina. Clinical phenotypes are diverse, ranging from mild night blindness to severe visual impairments. There is currently no cure for RHO-adRP. Although there have been significant advances in gene therapy for inherited retinal diseases, treating RHO-adRP presents a unique challenge since it is an autosomal dominant disease caused by more than 150 gain-of-function mutations in the RHO gene, rendering the established gene supplementation strategy inadequate. This review provides an update on RNA therapeutics and therapeutic editing genome surgery strategies and ongoing clinical trials for RHO-adRP, discussing mechanisms of action, preclinical data, current state of development, as well as risk and benefit considerations. Potential outcome measures useful for future clinical trials are also addressed. Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP) is a hereditary degenerative disorder in which mutations in the gene encoding RHO, the light-sensitive G protein-coupled receptor involved in phototransduction in rods, lead to progressive loss of rods and subsequently cones in the retina. Clinical phenotypes are diverse, ranging from mild night blindness to severe visual impairments. There is currently no cure for RHO-adRP. Although there have been significant advances in gene therapy for inherited retinal diseases, treating RHO-adRP presents a unique challenge since it is an autosomal dominant disease caused by more than 150 gain-of-function mutations in the RHO gene, rendering the established gene supplementation strategy inadequate. This review provides an update on RNA therapeutics and therapeutic editing genome surgery strategies and ongoing clinical trials for RHO-adRP, discussing mechanisms of action, preclinical data, current state of development, as well as risk and benefit considerations. Potential outcome measures useful for future clinical trials are also addressed. Retinitis pigmentosa (RP) is a group of rare inherited disorders with phenotypes ranging from mild night blindness (nyctalopia) to total blindness, affecting 1 in 3,000–7,000 individuals.1Tsang S.H. Sharma T. Retinitis pigmentosa (non-syndromic).Adv. Exp. Med. Biol. 2018; 1085: 125-130Crossref PubMed Scopus (21) Google Scholar,2Verbakel S.K. van Huet R.A.C. Boon C.J.F. den Hollander A.I. Collin R.W.J. Klaver C.C.W. Hoyng C.B. Roepman R. Klevering B.J. Non-syndromic retinitis pigmentosa.Prog. Retin. Eye Res. 2018; 66: 157-186Crossref PubMed Scopus (219) Google Scholar Aberrations in photoreceptors (rods and cones) and the retinal pigment epithelium cause progressive vision loss. In the initial stages of the disease process, rod photoreceptors start to die, and this causes night blindness. Affected individuals begin to experience difficulty seeing in dim light and adapting to changes in light sensitivity, leading to difficulties with driving at night or entering darkened rooms. Following the loss of rod photoreceptors, cone photoreceptors (which produce high acuity, bright light vision) are progressively lost. The cone degeneration in RP is considered secondary to rod death, possibly due to neighbor effects of decreased trophic factors, nutrient shortage, and oxidative stress.3Narayan D.S. Wood J.P. Chidlow G. Casson R.J. A review of the mechanisms of cone degeneration in retinitis pigmentosa.Acta Ophthalmol. 2016; 94: 748-754Crossref PubMed Scopus (81) Google Scholar,4Caruso S. Ryu J. Quinn P.M. Tsang S.H. Precision metabolome reprogramming for imprecision therapeutics in retinitis pigmentosa.J. Clin. Invest. 2020; 130: 3971-3973PubMed Google Scholar The progression of RP is slow and involves the continuous loss of photoreceptors, leading to loss of peripheral vision termed “tunnel vision,” whereby only the central vision is preserved. RP patients also report experiencing continuous flashes of light (photopsia). In late stages of the disease central vision is lost, resulting in total blindness. Patients may lose up to 90% of rod cells before vision changes are detected, resulting in initial diagnosis at advanced stages of disease.5Malanson K.M. Lem J. Rhodopsin-mediated retinitis pigmentosa.Prog. Mol. Biol. Transl. Sci. 2009; 88: 1-31Crossref PubMed Scopus (32) Google Scholar An electroretinogram (ERG) evaluating rod and cone functions can detect changes in rod function during initial stages of the disease before significant visual dysfunctions occur.6Gouras P. Carr R.E. Electrophysiological studies in early retinitis pigmentosa.Arch. Ophthalmol. 1964; 72: 104-110Crossref PubMed Scopus (39) Google Scholar,7Berson E.L. Retinitis pigmentosa and allied diseases: applications of electroretinographic testing.Int. Ophthalmol. 1981; 4: 7-22Crossref PubMed Scopus (45) Google Scholar The rhodopsin (RHO) gene was the first identified gene causing RP.8Dryja T.P. McGee T.L. Reichel E. Hahn L.B. Cowley G.S. Yandell D.W. Sandberg M.A. Berson E.L. A point mutation of the rhodopsin gene in one form of retinitis pigmentosa.Nature. 1990; 343: 364-366Crossref PubMed Scopus (755) Google Scholar,9Dryja T.P. McGee T.L. Hahn L.B. Cowley G.S. Olsson J.E. Reichel E. Sandberg M.A. Berson E.L. Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa.N. Engl. J. Med. 1990; 323: 1302-1307Crossref PubMed Scopus (327) Google Scholar Human RHO contains five exons and is 6.7 kb, and it is located on chromosome 3q22.1.10National Center for Biotechnology InformationGene. RHO rhodopsin [Homo sapiens (human)].2020https://www.ncbi.nlm.nih.gov/gene/6010Google Scholar More than 150 different mutations in RHO are associated with 25% of autosomal dominantly inherited RP (adRP) cases. Patients with RHO-mediated adRP have discernible differences in the pattern of retinal dysfunction between families with different mutations.11Jacobson S.G. Kemp C.M. Sung C.H. Nathans J. Retinal function and rhodopsin levels in autosomal dominant retinitis pigmentosa with rhodopsin mutations.Am. J. Ophthalmol. 1991; 112: 256-271Abstract Full Text PDF PubMed Scopus (95) Google Scholar Two classes of RHO-adRP have been described in the literature based on clinical observations. Class A patients (possessing mutations R135G, R135L, R135W, V345L, and P347L) lose rod function over the entirety of the retina and experience onset of night blindness earlier in life.12Cideciyan A.V. Hood D.C. Huang Y. Banin E. Li Z.Y. Stone E.M. Milam A.H. Jacobson S.G. Disease sequence from mutant rhodopsin allele to rod and cone photoreceptor degeneration in man.Proc. Natl. Acad. Sci. USA. 1998; 95: 7103-7108Crossref PubMed Scopus (215) Google Scholar There is a catastrophic loss of rod function, which may not be corrected, and therefore therapies should be focused on cone preservation in these patients. Class B patients demonstrate a milder phenotype, including normal rod activation kinetics and preserved rod outer segment length with abnormalities in the rod visual cycle that are mutation specific. Among subclass B1 (T17M, P23H, T58R, V87D, G106R, and D190G) patients, photoreceptor degeneration is heterogeneous and patients show an inferior to superior disease progression. Subclass B2 patients (G51A, Q64ter, and Q344ter) show no regional retinal predisposition for disease. In class B patients, rods have the potential to be rescued, and rod preservation should be a target in order to protect cones. Of note, the P23H mutation is the most prevalent RHO mutation in North America, accounting for 10% of adRP cases because of a founder effect. P23H is not found elsewhere, including Europe and Asia. Individuals possessing the P23H mutation have significantly better visual acuity and larger electroretinographic amplitudes.13Berson E.L. Rosner B. Sandberg M.A. Dryja T.P. Ocular findings in patients with autosomal dominant retinitis pigmentosa and a rhodopsin gene defect (Pro-23-His).Arch. Ophthalmol. 1991; 109: 92-101Crossref PubMed Scopus (145) Google Scholar Multiple mechanisms underlie RHO-mediated retinal degeneration.14Athanasiou D. Aguila M. Bellingham J. Li W. McCulley C. Reeves P.J. Cheetham M.E. The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy.Prog. Retin. Eye Res. 2018; 62: 1-23Crossref PubMed Scopus (110) Google Scholar RHO is the visual pigment of retinal rods, which facilitates vision in dim light and absorbs light at 495 nm.15Nathans J. Piantanida T.P. Eddy R.L. Shows T.B. Hogness D.S. Molecular genetics of inherited variation in human color vision.Science. 1986; 232: 203-210Crossref PubMed Scopus (508) Google Scholar It is a 348-aa G protein-coupled receptor protein with seven transmembrane domains, with a luminal N terminus and a cytoplasmic C terminus.16Nathans J. Hogness D.S. Isolation and nucleotide sequence of the gene encoding human rhodopsin.Proc. Natl. Acad. Sci. USA. 1984; 81: 4851-4855Crossref PubMed Scopus (393) Google Scholar The cytoplasmic face of RHO is made of three loops with catalytic sites that prompt guanosine triphosphate (GTP)-guanosine diphosphate GDP exchange by transducing GNAT1 and sites for light-dependent phosphorylation by RHO kinase. It also contains sites sites for N-glycosylation, and the site lys296 is where retinal attachment occurs. The vast majority of pathogenic mutations in RHO cause retinal degeneration by way of gain-of-function mutations leading to adRP. Of note, a few RHO mutations have been associated with autosomal recessive RP, but they are relatively uncommon.14Athanasiou D. Aguila M. Bellingham J. Li W. McCulley C. Reeves P.J. Cheetham M.E. The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy.Prog. Retin. Eye Res. 2018; 62: 1-23Crossref PubMed Scopus (110) Google Scholar Further details regarding the molecular and cellular basis of RHO-mediated RP are beyond the scope of this review and can be found in the excellent review by Athanasiou et al.14Athanasiou D. Aguila M. Bellingham J. Li W. McCulley C. Reeves P.J. Cheetham M.E. The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy.Prog. Retin. Eye Res. 2018; 62: 1-23Crossref PubMed Scopus (110) Google Scholar There is no universally effective treatment or cure for RHO-RP, and multiple approaches are being studied. Stem cell or retinal tissue transplantation, nutritional supplementations, retinal implants, as well as targeted and non-targeted gene therapies have been proposed and tested. Transplantation of stem cells or retinal tissue by the use of retinal progenitor cells may provide beneficial effects. In studies that tested the effect of transplanted newly born rods, responses to dim light were restored in blind mice.17MacLaren R.E. Pearson R.A. MacNeil A. Douglas R.H. Salt T.E. Akimoto M. Swaroop A. Sowden J.C. Ali R.R. Retinal repair by transplantation of photoreceptor precursors.Nature. 2006; 444: 203-207Crossref PubMed Scopus (836) Google Scholar Nutritional supplementation for RP with nutrients such as vitamin A, docosahexaenoic acid (DHA), and lutein have produced limited and controversial results.18Berson E.L. Rosner B. Sandberg M.A. Hayes K.C. Nicholson B.W. Weigel-DiFranco C. Willett W. A randomized trial of vitamin A and vitamin E supplementation for retinitis pigmentosa.Arch. Ophthalmol. 1993; 111: 761-772Crossref PubMed Scopus (452) Google Scholar Retinal implants and prostheses have been investigated as potential interventions in the treatment of advanced RP, including the Argus II retinal prosthesis system and the Alpha IMS from Retina Implant. Clinical trials showed improvement in various visual function tests but several serious adverse events.19da Cruz L. Dorn J.D. Humayun M.S. Dagnelie G. Handa J. Barale P.O. Sahel J.A. Stanga P.E. Hafezi F. Safran A.B. et al.Argus II Study GroupFive-year safety and performance results from the Argus II Retinal Prosthesis System clinical trial.Ophthalmology. 2016; 123: 2248-2254Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,20Wang A.L. Knight D.K. Vu T.T. Mehta M.C. Retinitis pigmentosa: review of current treatment.Int. Ophthalmol. Clin. 2019; 59: 263-280Crossref PubMed Scopus (19) Google Scholar Optogenetics delivered as non-targeted gene therapy for advanced RP are also being tested. Channelrhodopsins (ChRs), when expressed in the retina, depolarize in response to light-generating signals, which are then transmitted to the brain. There are two ongoing current clinical trials using optogenetics in RP patients, RST-001 (ClinicalTrials.gov: NCT02556736) and GS030 (ClinicalTrials.gov: NCT03326336). Targeted gene therapy holds great promise for RHO-adRP and is the focus of this review. There have been recent advances in gene replacement therapies for autosomal recessive and X-linked inherited retinal disorders. In these cases, since the genes of interest have loss-of-function mutations (both copies in autosomal recessive disorders and the only copy in X-linked disorders), a straightforward replacement approach by gene supplementation is appropriate. The first, and currently only, US Food and Drug Administration (FDA)-approved gene therapy for a retinal disease is voretigene neparvovec-rzyl (Luxturna), a gene replacement therapy targeting RPE65 enzyme deficiency in Leber congenital amaurosis (LCA) and RP. Subretinal injection of the adeno-associated viruses (AAVs)-hRPE65 vector expressing RPE65 restores its production in the transduced RPE cells, resulting in functional visual improvements measured by navigational ability and light sensitivity.21Maguire A.M. Russell S. Wellman J.A. Chung D.C. Yu Z.F. Tillman A. Wittes J. Pappas J. Elci O. Marshall K.A. et al.Efficacy, safety, and durability of voretigene neparvovec-rzyl in RPE65 mutation-associated inherited retinal dystrophy: results of phase 1 and 3 trials.Ophthalmology. 2019; 126: 1273-1285Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar In addition, there are many ongoing clinical trials with gene supplementation therapies for X-linked RP caused by RP GTPase regulator (RPGR) mutations, autosomal recessive RP caused by PDE6B and MERTK mutations, autosomal recessive achromatopsia caused by CNGA3 and CNGB3 mutations, X-linked choroideremia caused by REP1 mutations, as well as X-linked retinoschisis caused by RS1 mutations (https://clinicaltrials.gov/). In contrast, treating autosomal dominant diseases with gain-of-function mutations such as RHO-adRP requires a substantially different approach and has been traditionally difficult. The goal for targeted gene therapy of RHO-mediated adRP is to inhibit expression of the mutant RHO protein and to increase the ratio of wild-type (WT)-to-mutant RHO in order to reduce the rate of retinal degeneration. One approach is to design therapeutics with high specificity that can differentiate between a single point mutation between the diseased and WT RHO alleles in order to selectively decrease expression of toxic protein from mutant RHO. Although it is preferable to leave the WT RHO intact, gene therapy strategies that offer such a high level of allele selectivity have to be designed specifically for each of the 150+ mutations in RHO, making it impractical. An alternative approach involves disrupting both copies of endogenous RHO genes, mutant and WT, and replacing them with an exogenous RHO gene. Such a strategy can be mutation-independent and bypass the need to design unique therapy for each RHO mutant, offering a simplified treatment strategy for all RHO-adRP patients. Herein, we provide a review of both mutation-specific and mutation-independent gene therapy strategies currently under development for RHO-adRP (Figure 1; Table 1). For the purpose of this review, gene therapy is defined as delivering nucleic acids in vivo and covers both RNA and DNA targeting therapeutics.Table 1Comparison of Gene Therapy Candidates Currently under Development for RHO-adRPDeveloperDrug NameTargeted MutationMechanism of ActionPreclinical Model and Efficacy DataRoute of Administration/ FrequencyCurrent Stage of DevelopmentRNA targetingProQR Therapeutics/Ionis PharmaceuticalsPR1123/ION357P23Hantisense oligonucleotides targeting mutated RHO mRNA• murine/rat• intravitreal injections• phase 1/2 started October 2019• 40% knockdown of RHO expression; ONL 18% thicker; ERG amplitude 181% higher (scotopic a-wave)• expected to need repeat injections• target to enroll 35 patients• expected to conclude in October 2021IVERIC BioIC-100mutation-independentshRNA suppression of endogenous RHO expression + replacement with codon-modified shRNA-resistant RHO delivered by a single AAV vector• caninesingle subretinal injectionplan to initiate a phase 1/2 clinical trial during the fourth quarter of 2020• >97% knockdown of RHO expression; qualitative ONL and ERG improvement• estimated equivalent human years of useful vision gain: 8aEstimation based on extrapolation of original data from preclinical studies. There is significant variability in the models and outcome measures used across studies.Roche/Spark TherapeuticsRhoNovamutation-independentshRNA suppression of endogenous RHO expression + replacement with codon-modified shRNA-resistant RHO delivered by dual AAV vectors• murinesingle subretinal injection• preclinical• 68% knockdown of RHO expression; ONL 35% thicker; ERG amplitude 244%–429% higher (rod-isolated response)• received orphan drug designations in Europe (2010) and in the US (2013)• estimated equivalent human years of useful vision gain: 11aEstimation based on extrapolation of original data from preclinical studies. There is significant variability in the models and outcome measures used across studies.Alnylam/Regeneron––RNAi molecule targeting RHO––• preclinicalTherapeutic editing genome surgeryEditas Medicine–mutation-independentCRISPR-based knockout of endogenous RHO genes + replacement RHO cDNA delivered by dual AAV vectors• in vitro (preliminary)single subretinal injection (presumed)• preclinical• 70% knockdown of RHO expression• declared development candidates May 2020Precision Bio–P23HARCUS meganuclease-medicated knockout of mutant RHO delivered by a single AAV vector• swine (preliminary)single subretinal injection• preclinical• qualitative rescue of outer retinal morphology and ERG rod response• estimated equivalent human years of useful vision gain: 14.5aEstimation based on extrapolation of original data from preclinical studies. There is significant variability in the models and outcome measures used across studies.a Estimation based on extrapolation of original data from preclinical studies. There is significant variability in the models and outcome measures used across studies. Open table in a new tab ASOs are single-stranded DNA molecules complementary to mRNA targets. Upon hybridization with target RNA through specific nucleotide pairing, ASOs induce target RNA degradation by recruiting cellular enzyme ribonuclease H1 (RNase H1), which cleaves the target RNA.22Crooke S.T. Molecular mechanisms of antisense oligonucleotides.Nucleic Acid Ther. 2017; 27: 70-77Crossref PubMed Scopus (138) Google Scholar ASOs remain intact through this process and therefore can be active for additional targets. This approach has been routinely used in basic research to achieve downregulation of gene expression. Due to recent advances in ASO technology, it is now possible for second-generation ASOs to selectively target a mutant allele with a single base pair difference from the WT allele, significantly expanding their potential as disease-modifying therapeutics for autosomal dominant genetic disorders in vivo.23Scoles D.R. Minikel E.V. Pulst S.M. Antisense oligonucleotides: a primer.Neurol. Genet. 2019; 5: e323Crossref PubMed Scopus (54) Google Scholar Three ASO medications have been successfully commercialized to date, including Spinraza/nusinersen for spinal muscular atrophy, Tegsedi/inotersen for hereditary transthyretin-mediated amyloidosis, and Waylivra/volanesorsen for familial chylomicronemia syndrome. Dozens more ASO drug candidates are currently under clinical development covering a broad range of disease areas. PR1123 (previously named ION357) is an ASO drug in a gapmer configuration targeting the P23H mutation in the human RHO gene. It has been shown to knock down expression of P23H mutant mRNA specifically without affecting the WT RHO RNA expression in cell lines and mouse models.24Biasutto P. Adamson P.S. Dulla K. Murray S. Monia B. McCaleb M. Allele specific knock-down of human P23H rhodopsin mRNA and prevention of retinal degeneration in humanized P23H rhodopsin knock-in mouse, following treatment with an intravitreal GAPmer antisense oligonucleotide (QR-1123).Invest. Ophthalmol. Vis. Sci. 2019; 60: 5719Google Scholar Unilateral intravitreal injection of PR1123 caused a 40% reduction in the RHO mRNA level compared to the contralateral eye in P23H mice while no significant change in RHO level was observed in WT mice. Moreover, PR1123 treatment led to ERG improvement and structural preservation in P23H mouse and rat models.24Biasutto P. Adamson P.S. Dulla K. Murray S. Monia B. McCaleb M. Allele specific knock-down of human P23H rhodopsin mRNA and prevention of retinal degeneration in humanized P23H rhodopsin knock-in mouse, following treatment with an intravitreal GAPmer antisense oligonucleotide (QR-1123).Invest. Ophthalmol. Vis. Sci. 2019; 60: 5719Google Scholar,25Murray S.F. Jazayeri A. Matthes M.T. Yasumura D. Yang H. Peralta R. Watt A. Freier S. Hung G. Adamson P.S. et al.Allele-specific inhibition of rhodopsin with an antisense oligonucleotide slows photoreceptor cell degeneration.Invest. Ophthalmol. Vis. Sci. 2015; 56: 6362-6375Crossref PubMed Scopus (50) Google Scholar Improved scotopic a-wave response amplitude at all stimulus intensities were observed via ERG on P23H rats following treatment. Treated eyes of P23H mice and rats had increased outer nuclear layer (ONL) thickness, a measurement of photoreceptor cell number. Additionally, a safety study was performed in primates that showed that QR-1123 did not affect levels of WT RHO mRNA in cynomolgus monkeys after single intravitreal injections. Initially developed by Ionis Pharmaceuticals, PR1123 was in-licensed to ProQR Therapeutics in 2018. It received orphan drug and fast track designations for P23H-adRP by the FDA in 2019. ProQR Therapeutics is currently sponsoring a phase 1/2 study that started recruitment in October 2019 (ClinicalTrials.gov: NCT04123626). The study intends to enroll up to 35 adult P23H-adRP patients followed for 12 months. An open label single-dose cohort as well as a double-masked multiple-dose escalation cohort are planned with repeat unilateral intravitreal injection of QR-1123 every 3 months compared to unilateral sham procedures. In addition to evaluating safety and tolerability as primary outcome measures, multiple modalities will be used to evaluate efficacy by measuring structural and functional improvements. The phase 1/2 study is scheduled to conclude in October 2021. shRNAs are short RNA molecules that spontaneously form hairpin structures. shRNAs are recognized by the intracellular RNA interference (RNAi) pathway, such as RNase III enzyme Dicer, and are processed to active small interfering RNAs (siRNAs). Subsequently, the siRNAs bind to target mRNAs through base pairing facilitated by the RNA-induced silencing complex, resulting in cleavage and degradation of target mRNAs. shRNAs are expressed by DNA vectors in contrast to their synthetic siRNA counterparts.26Lambeth L.S. Smith C.A. Short hairpin RNA-mediated gene silencing.Methods Mol. Biol. 2013; 942: 205-232Crossref PubMed Scopus (56) Google Scholar In vivo delivery of DNA vectors expressing shRNAs often requires viral vectors such as AAV and lentiviruses. A dual AAV vector strategy of suppression with shRNA and replacement was tested in a murine model of RHO-adRP, Rhop347s/+, by Millington-Ward et al.27Millington-Ward S. Chadderton N. O’Reilly M. Palfi A. Goldmann T. Kilty C. Humphries M. Wolfrum U. Bennett J. Humphries P. et al.Suppression and replacement gene therapy for autosomal dominant disease in a murine model of dominant retinitis pigmentosa.Mol. Ther. 2011; 19: 642-649Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar in a proof-of-concept study.28Kiang A.S. Palfi A. Ader M. Kenna P.F. Millington-Ward S. Clark G. Kennan A. O’reilly M. Tam L.C. Aherne A. et al.Toward a gene therapy for dominant disease: validation of an RNA interference-based mutation-independent approach.Mol. Ther. 2005; 12: 555-561Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar One AAV2/5 vector, AAV-shRNA, expresses an shRNA targeting human RHO gene at nucleotides 254–274, a site independent of known mutations. Therefore, it is designed to knock down expression of all endogenous RHO genes, mutant and WT, in a mutation-independent way.29O’Reilly M. Palfi A. Chadderton N. Millington-Ward S. Ader M. Cronin T. Tuohy T. Auricchio A. Hildinger M. Tivnan A. et al.RNA interference-mediated suppression and replacement of human rhodopsin in vivo.Am. J. Hum. Genet. 2007; 81: 127-135Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar An shRNA-resistant human RHO with codon modification at wobble nucleotides is expressed by a second AAV2/5 vector as replacement (AAV-RHO). Of note, the endogenous murine Rho gene is naturally resistant to the shRNA due to mismatches at the RNAi target site between the two species and was continuously expressed in this model. Both vectors were delivered together through subretinal injections to Rhop347s/+ mice. An AAV vector expressing non-targeting control RNAi was used as a control. A 68% ± 2.4% reduction in the ratio of RHO/Rho mRNA was observed in transduced retinal cells after AAV-shRNA injection, suggesting an efficient knockdown of RHO gene expression. As for replacement, AAV-RHO injection in WT mice resulted in RHO/Rho ratio of 31% ± 5% at the mRNA level. The authors argued that since only 40% of the retina was transduced but the whole retina was analyzed, the true expression level of AAV-RHO in transduced cells could be much higher. To evaluate functional improvement, rod-mediated ERG responses were analyzed. Rod b-wave amplitudes were 184.5 ± 65.4 μV in treated eyes at 6 weeks post-injection (wpi) compared to control eyes at 34.9 ± 16.8 μV. The improvement was still significant at 20 wpi (58.1 ± 19.8 μV in the dual vector group versus 16.9 ± 12.6 μV in the control vector group). The control single vector injections did not result in improvement in rod responses. Histologically, ONL thickness was 17.9 ± 3.4 μm in dual vector-injected eyes compared to 13.3 ± 2.0 μm in sections from control eyes at 6 wpi. At 20 wpi, ONL thickness was 8.9 ± 1.2 μm in treated eyes, whereas in control eyes the ONL had almost completely disappeared and was no longer measurable due to disease progression. Additionally, electron microscopy revealed preservation of rod photoreceptor outer segments with correctly formed membrane disks in treated eyes compared to control eyes, which exhibited degenerating outer segments and disorganized membrane disks. This dual vector shRNA suppression and replacement therapeutic strategy for RHO-adRP was named RhoNova and received orphan drug designation in Europe in 2010 and in the US in 2013. Genable Technologies, the original owner of RhoNova, was acquired by Spark Therapeutics in 2016, which was subsequently acquired by Roche in 2019. There has been no publicly available update on RhoNova’s clinical development. More recently, a gene therapy candidate for RHO-adRP composed of a single AAV2/5 vector expressing both an shRNA targeting human RHO and a healthy copy of the RHO gene modified to be resistant to the shRNA has been developed.30Cideciyan A.V. Sudharsan R. Dufour V.L. Massengill M.T. Iwabe S. Swider M. Lisi B. Sumaroka A. Marinho L.F. Appelbaum T. et al.Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector.Proc. Natl. Acad. Sci. USA. 2018; 115: E8547-E8556Crossref PubMed Scopus (62) Google Scholar The shRNA targets a part of human RHO unaffected by any known mutations, similar to RhoNova. A codon-modified human RHO gene resistant to shRNA is expressed by the same vector as replacement. Cideciyan et al.30Cideciyan A.V. Sudharsan R. Dufour V.L. Massengill M.T. Iwabe S. Swider M. Lisi B. Sumaroka A. Marinho L.F. Appelbaum T. et al.Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector.Proc. Natl. Acad. Sci. USA. 2018; 115: E8547-E8556Crossref PubMed Scopus (62) Google Scholar performed preclinical studies in a naturally occurring canine model of RHO-adRP (RHOT4R/+) sensitive to acute light-induced retinal degeneration with single subretinal injections. One of the shRNA candidates, shRNA820, caused a near-complete knockdown of RHO mRNA and protein in WT and RHO mutant retinas. When the shRNA-resistant human RHO gene, named RHO820, was supplemented in addition to shRNA820 by a single AAV2/5 viral vector, the resistant RHO was expressed at 118%–132% at the mRNA level and 31%–33% at the protein level compared to untreated retinas. There was some evidence that this suppression and replacement strategy could rescue the light-induced retinal degeneration in RHO mutant retina. ERG measurements showed greater rod and cone responses in AAV-shRNA820-RHO820-injected retinas compared to those injected with balanced salt solution. There were also qualitative data suggesting retention of ONL thickness, photoreceptor cell bo" @default.
• W3080180587 created "2020-09-01" @default.
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• W3080180587 creator A5030588350 @default.
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• W3080180587 date "2020-10-01" @default.
• W3080180587 modified "2023-10-16" @default.
• W3080180587 title "Therapy in Rhodopsin-Mediated Autosomal Dominant Retinitis Pigmentosa" @default.
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• W3080180587 doi "https://doi.org/10.1016/j.ymthe.2020.08.012" @default.
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