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Shannon M. McNamee , Natalie P. Chan , Monica Akula , Marielle O. Avola , Maiya Whalen , Kaden Nystuen , Pushpendra Singh , Arun K. Upadhyay , Margaret M. DeAngelis , Neena B. Haider | Gene Therapy | 26 January 2024 | Vol 31 | Pgs. 255–262 | https://doi.org/10.1038/s41434-024-00440-6 Abstract Retinitis pigmentosa (RP) is a heterogeneous disease and the main cause of vision loss within the group of inherited retinal diseases (IRDs). IRDs are a group of rare disorders caused by mutations in one or more of over 280 genes which ultimately result in blindness. Modifier genes play a key role in modulating disease phenotypes, and mutations in them can affect disease outcomes, rate of progression, and severity. Our previous studies have demonstrated that the nuclear hormone receptor 2 family e, member 3 ( Nr2e3 ) gene reduced disease progression and loss of photoreceptor cell layers in RhoP23H − / − mice. This follow up, pharmacology study evaluates a longitudinal NR2E3 dose response in the clinically relevant heterozygous RhoP23H mouse. Reduced retinal degeneration and improved retinal morphology was observed 6 months following treatment evaluating three different NR2E3 doses. Histological and immunohistochemical analysis revealed regions of photoreceptor rescue in the treated retinas of RhoP23H+/ − mice. Functional assessment by electroretinogram (ERG) showed attenuated photoreceptor degeneration with all doses. This study demonstrates the effectiveness of different doses of NR2E3 at reducing retinal degeneration and informs dose selection for clinical trials of RhoP23H -associated RP. Introduction Retinitis pigmentosa (RP) is the most common form of inherited blindness. RP is a heterogeneous disease that varies in age of onset, rate of progression and genetic etiology based on the mutation and gene impacted [ 1 , 2 , 3 , 4 , 5 ]. Retinitis pigmentosa affects 1 in 4000 individuals worldwide [ 6 , 7 ]. RP diseases include syndromic and non-syndromic forms with over 200 unique genetic mutations associated with disease onset [ 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. There is currently only one type of treatment approved to treat a specific form of RP, voretigene neparvovec (Luxturna®) is approved for treatment in patients with biallelic RPE65 gene mutations [ 15 , 16 , 17 , 18 ]. Additionally, over 40% of RP cases cannot be genetically diagnosed [ 19 ]. While there is great heterogeneity in RP disease, the common shared pathology is degeneration of photoreceptor (PR) cells. The genes and mechanisms causing photoreceptor degeneration can vary and the cumulative degenerative outcome is influenced by a mutational load on the system that includes the primary mutation and modifier genes among other factors [ 20 ]. Our previous publication revealed the novel finding that the modifier gene Nr2e3 can treat retinal degeneration in several mouse models of RP [ 19 ]. Modifier genes are defined as allelic variants within a normal population, and can significantly impact the onset, progression, and severity of diseases [ 21 , 22 , 23 , 24 ]. Studies of multiple diseases including spinal muscular atrophy, spinocerebellar ataxia type 1, dystonia, epileptic encephalopathy, cystic fibrosis, and retinal degeneration show the effect of modifier genes and their impact on disease phenotypes altered by shifted genetic backgrounds [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ]. Mutations in the modifier gene nuclear hormone receptor NR2E3 are associated with several types of retinal degeneration including clumped pigmentary retinal degeneration (CPRD), Goldmann–Favre syndrome (GFS), enhanced S-cone syndrome (ESCS), and autosomal dominant retinitis pigmentosa (adRP) [ 34 ]. The variable phenotypes of NR2E3 -associated retinal degeneration suggest modifier genes could be influencing disease manifestation and outcomes. Nr2e3 plays a major role in the retina by regulating the development and maintenance of photoreceptor cells, and regulating gene networks in pathways including ER stress, neuroprotection, photoreceptor function, apoptosis, immune response, and cell survival, and thereby impacting homeostasis of the retina [ 19 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ]. Modifier genes such as NR2E3 could thus be a more effective therapeutic agent and especially beneficial in RP cases where the primary mutation cannot be identified. Previous publications by our lab examined the effectiveness of Nr2e3 as a therapeutic for NR2E3 -associated RP as well as other forms of RP that do not possess an NR2E3 mutation [ 19 ]. Although RP is a rare disease, mutations in the Rhodopsin gene represent about 40% of all adRP cases [ 42 ]. Additionally, there are more than 150 known mutations in Rhodopsin and the RhoP23H mutation accounts for about 10% of Rho related adRP in the United States alone [ 42 , 43 , 44 , 45 ]. The RhoP23H mutation creates a misfolded protein that is not processed properly and accumulates in the endoplasmic reticulum (ER) [ 19 , 45 ]. The accumulation of these aberrant Rho proteins causes cellular stress [ 43 , 46 , 47 , 48 ]. Patients with this mutation usually experience night-blindness followed by a progressive loss of their visual field [ 42 , 43 , 44 , 45 , 46 , 47 , 48 ]. This was a pharmacological study to demonstrate the efficacy of dosage and longitudinal impact of NR2E3 modifier gene therapy to treat retinal degeneration in the RhoP23H+/ − mouse when administered during early/intermediate degeneration. Treated animals were assessed 1, 3, and 6 months after dose administration to track the degree of longitudinal rescue of degeneration. The RhoP23H mouse is a model for human Retinitis Pigmentosa 4 (RP-4). These mice harbor a substitution of the amino acid proline with histidine at position 23 resulting in protein misfolding and degradation from an aberrant message [ 19 , 45 ]. Heterozygous RhoP23H mice were used for this study, as they are more clinically relevant with adRP features including gradual loss of rods and scotopic ERG function followed by loss of cones and photopic ERG function [ 45 , 49 , 50 ]. RhoP23H+/ − mice exhibit rapid photoreceptor degeneration from postnatal day (P) 15 to P30 that progresses to gradual degeneration over time [ 45 , 49 , 50 ]. The outer nuclear layer (ONL) thickness of a P30 RhoP23H+/ − retina is about 30–40% less than a normal wild-type retina and continues to gradually decrease with age [ 45 , 49 ]. RhoP23H+/ − mice retain a majority of their photoreceptors; however, the cells in the inner and outer segments show structural disorganization and as mice age the outer segments (OS) become shorter [ 44 ]. P35 mice possess shorter rod OS and by P63, have about half the normal amount of rod nuclei compared to wild-type mice [ 45 ]. Scotopic ERG responses are severely reduced by P41 and are nearly undetectable by P170 [ 45 ]. In comparison, homozygous mice exhibit severe rapid retinal degeneration by P23 with almost complete loss of photoreceptors by P63 [ 45 ]. It is important to note, our previous study of Nr2e3 therapeutic utilized RhoP23H − / − mice which possess an abnormal fundus phenotype whereas this study utilized RhoP23H+/ − mice that have a normal fundus phenotype [ 19 ]. This study demonstrates that low, mid, and high doses of AAV5- hNR2E3 can slow retinal degeneration in the RhoP23H+/ − mouse for at least 6 months following treatment when administered during early/intermediate disease. Click here to read entire paper References Hamel C. Retinitis pigmentosa. Orphanet J Rare Dis. 2006;1:40. al-Maghtheh M, Inglehearn CF, Keen TJ, Evans K, Moore AT, Jay M, et al. Identification of a sixth locus for autosomal dominant retinitis pigmentosa on chromosome 19. Hum Mol Genet. 1994;3:351–4. Andréasson S, Ponjavic V, Abrahamson M, Ehinger B, Wu W, Fujita R, et al. Phenotypes in three Swedish families with X-linked retinitis pigmentosa caused by different mutations in the RPGR gene. Am J Ophthalmol. 1997;124:95–102. Blanton SH, Heckenlively JR, Cottingham AW, Friedman J, Sadler LA, Wagner M, et al. Linkage mapping of autosomal dominant retinitis pigmentosa (RP1) to the pericentric region of human chromosome 8. Genomics. 1991;11:857–69. Ali MU, Rahman MSU, Cao J, Yuan PX. Genetic characterization and disease mechanism of retinitis pigmentosa; current scenario. 3 Biotech. 2017;7:251.   Ferrari S, Di Iorio E, Barbaro V, Ponzin D, Sorrentino FS, Parmeggiani F. Retinitis pigmentosa: genes and disease mechanisms. Curr Genomics. 2011;12:238–49. Daiger SP, Sullivan LS, Bowne SJ. Genes and mutations causing retinitis pigmentosa. Clin Genet. 2013;84:132–41. Bunker CH, Berson EL, Bromley WC, Hayes RP, Roderick TH. Prevalence of retinitis pigmentosa in Maine. Am J Ophthalmol. 1984;97:357–65. Grøndahl J. Estimation of prognosis and prevalence of retinitis pigmentosa and Usher syndrome in Norway. Clin Genet. 1987;31:255–64.   Pierrottet CO, Zuntini M, Digiuni M, Bazzanella I, Ferri P, Paderni R, et al. Syndromic and non-syndromic forms of retinitis pigmentosa: a comprehensive Italian clinical and molecular study reveals new mutations. Genet Mol Res. 2014;13:8815–33. Ahmed ZM, Riazuddin S, Riazuddin S, Wilcox ER. The molecular genetics of Usher syndrome. Clin Genet. 2003;63:431–44. Boughman JA, Vernon M, Shaver KA. Usher syndrome: definition and estimate of prevalence from two high-risk populations. J Chronic Dis. 1983;36:595–603. Beales PL, Warner AM, Hitman GA, Thakker R, Flinter FA. Bardet-Biedl syndrome: a molecular and phenotypic study of 18 families. J Med Genet. 1997;34:92–8. Cox GF, Hansen RM, Quinn N, Fulton AB. Retinal function in carriers of Bardet-Biedl syndrome. Arch Ophthalmol. 2003;121:804–10. Cross N, van Steen C, Zegaoui Y, Satherley A, Angelillo L. Retinitis pigmentosa: burden of disease and current unmet needs. Clin Ophthalmol. 2022;16:1993–2010. Commissioner O of the. FDA. FDA. FDA approves novel gene therapy to treat patients with a rare form of inherited vision loss. 2020 [cited 2023 Sep 5]. Available from: https://www.fda.gov/news-events/press-announcements/fda-approves-novel-gene-therapy-treat-patients-rare-form-inherited-vision-loss . Miraldi Utz V, Coussa RG, Antaki F, Traboulsi EI. Gene therapy for RPE65-related retinal disease. Ophthalmic Genet. 2018;39:671–7. Kwak JJ, Kim HR, Byeon SH. Short-term outcomes of the first in vivo gene therapy for RPE65-mediated retinitis pigmentosa. Yonsei Med J. 2022;63:701–5. Li S, Datta S, Brabbit E, Love Z, Woytowicz V, Flattery K, et al. Nr2e3 is a genetic modifier that rescues retinal degeneration and promotes homeostasis in multiple models of retinitis pigmentosa. Gene Ther. 2021;28:223–41. Sohocki MM, Daiger SP, Bowne SJ, Rodriquez JA, Northrup H, Heckenlively JR, et al. Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies. Hum Mutat. 2001;17:42–51. Houlston RS, Tomlinson IP. Modifier genes in humans: strategies for identification. Eur J Hum Genet. 1998;6:80–8. Harper AR, Nayee S, Topol EJ. Protective alleles and modifier variants in human health and disease. Nat Rev Genet. 2015;16:689–701. Chow CY, Kelsey KJP, Wolfner MF, Clark AG. Candidate genetic modifiers of retinitis pigmentosa identified by exploiting natural variation in Drosophila. Hum Mol Genet. 2016;25:651–9. Haider NB, Ikeda A, Naggert JK, Nishina PM. Genetic modifiers of vision and hearing. Hum Mol Genet. 2002;11:1195–206. Hsieh CS, Macatonia SE, O’Garra A, Murphy KM. T cell genetic background determines default T helper phenotype development in vitro. J Exp Med. 1995;181:713–21.   Kiesewetter S, Macek M, Davis C, Curristin SM, Chu CS, Graham C, et al. A mutation in CFTR produces different phenotypes depending on chromosomal background. Nat Genet. 1993;5:274–8. Rose-Hellekant TA, Gilchrist K, Sandgren EP. Strain background alters mammary gland lesion phenotype in transforming growth factor-alpha transgenic mice. Am J Pathol. 2002;161:1439–47. Calhoun JD, Hawkins NA, Zachwieja NJ, Kearney JA. Cacna1g is a genetic modifier of epilepsy in a mouse model of Dravet syndrome. Epilepsia. 2017;58:e111–5. Eshraghi M, McFall E, Gibeault S, Kothary R. Effect of genetic background on the phenotype of the Smn2B/- mouse model of spinal muscular atrophy. Hum Mol Genet. 2016;25:4494–506. Tanabe LM, Martin C, Dauer WT. Genetic background modulates the phenotype of a mouse model of DYT1 dystonia. PLoS ONE. 2012;7:e32245. Ebermann I, Phillips JB, Liebau MC, Koenekoop RK, Schermer B, Lopez I, et al. PDZD7 is a modifier of retinal disease and a contributor to digenic Usher syndrome. J Clin Invest. 2010;120:1812–23. Maddox DM, Ikeda S, Ikeda A, Zhang W, Krebs MP, Nishina PM, et al. An allele of microtubule-associated protein 1A (Mtap1a) reduces photoreceptor degeneration in Tulp1 and Tub mutant mice. Invest Ophthalmol Vis Sci. 2012;53:1663–9. Fernandez-Funez P, Nino-Rosales ML, de Gouyon B, She WC, Luchak JM, Martinez P, et al. Identification of genes that modify ataxin-1-induced neurodegeneration. Nature. 2000;408:101–6. Schorderet DF, Escher P. NR2E3 mutations in enhanced S-cone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), clumped pigmentary retinal degeneration (CPRD), and retinitis pigmentosa (RP). Hum Mutat. 2009;30:1475–85. Cruz NM, Yuan Y, Leehy BD, Baid R, Kompella U, DeAngelis MM, et al. Modifier genes as therapeutics: the nuclear hormone receptor Rev Erb alpha (Nr1d1) rescues Nr2e3 associated retinal disease. PLoS ONE. 2014;9:e87942. Haider NB, Jacobson SG, Cideciyan AV, Swiderski R, Streb LM, Searby C, et al. Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate. Nat Genet. 2000;24:127–31. Sharon D, Sandberg MA, Caruso RC, Berson EL, Dryja TP. Shared mutations in NR2E3 in enhanced S-cone syndrome, Goldmann-Favre syndrome, and many cases of clumped pigmentary retinal degeneration. Arch Ophthalmol. 2003;121:1316–23. Coppieters F, Leroy BP, Beysen D, Hellemans J, De Bosscher K, Haegeman G, et al. Recurrent mutation in the first zinc finger of the orphan nuclear receptor NR2E3 causes autosomal dominant retinitis pigmentosa. Am J Hum Genet. 2007;81:147–57. Gire AI, Sullivan LS, Bowne SJ, Birch DG, Hughbanks-Wheaton D, Heckenlively JR, et al. The Gly56Arg mutation in NR2E3 accounts for 1-2% of autosomal dominant retinitis pigmentosa. Mol Vis. 2007;13:1970–5. Webber AL, Hodor P, Thut CJ, Vogt TF, Zhang T, Holder DJ, et al. Dual role of Nr2e3 in photoreceptor development and maintenance. Exp Eye Res. 2008;87:35–48. Cheng H, Aleman TS, Cideciyan AV, Khanna R, Jacobson SG, Swaroop A. In vivo function of the orphan nuclear receptor NR2E3 in establishing photoreceptor identity during mammalian retinal development. Hum Mol Genet. 2006;15:2588–602. Sudharsan R, Beltran WA. Progress in gene therapy for rhodopsin autosomal dominant retinitis pigmentosa. Adv Exp Med Biol. 2019;1185:113–8. Haeri M, Knox BE. Rhodopsin mutant P23H destabilizes rod photoreceptor disk membranes. PLoS ONE. 2012;7:e30101. Humphries MM, Rancourt D, Farrar GJ, Kenna P, Hazel M, Bush RA, et al. Retinopathy induced in mice by targeted disruption of the rhodopsin gene. Nat Genet. 1997;15:216–9. Sakami S, Maeda T, Bereta G, Okano K, Golczak M, Sumaroka A, et al. Probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to P23H opsin mutations. J Biol Chem. 2011;286:10551–67. Noorwez SM, Sama RRK, Kaushal S. Calnexin improves the folding efficiency of mutant rhodopsin in the presence of pharmacological chaperone 11-cis-retinal. J Biol Chem. 2009;284:33333–42. Noorwez SM, Kuksa V, Imanishi Y, Zhu L, Filipek S, Palczewski K, et al. Pharmacological chaperone-mediated in vivo folding and stabilization of the P23H-opsin mutant associated with autosomal dominant retinitis pigmentosa. J Biol Chem. 2003;278:14442–50. Noorwez SM, Malhotra R, McDowell JH, Smith KA, Krebs MP, Kaushal S. Retinoids assist the cellular folding of the autosomal dominant retinitis pigmentosa opsin mutant P23H. J Biol Chem. 2004;279:16278–84. Pasquale RL, Guo Y, Umino Y, Knox B, Solessio E. Temporal contrast sensitivity increases despite photoreceptor degeneration in a mouse model of retinitis pigmentosa. eNeuro. 2021;8:ENEURO.0020-21.2021. Chiang WC, Kroeger H, Sakami S, Messah C, Yasumura D, Matthes MT, et al. Robust endoplasmic reticulum-associated degradation of rhodopsin precedes retinal degeneration. Mol Neurobiol. 2015;52:679–95. Danciger M, Blaney J, Gao YQ, Zhao DY, Heckenlively JR, Jacobson SG, et al. Mutations in the PDE6B gene in autosomal recessive retinitis pigmentosa. Genomics. 1995;30:1–7. Mitra RN, Zheng M, Weiss ER, Han Z. Genomic form of rhodopsin DNA nanoparticles rescued autosomal dominant Retinitis pigmentosa in the P23H knock-in mouse model. Biomaterials. 2018;157:26–39. Mollema NJ, Yuan Y, Jelcick AS, Sachs AJ, von Alpen D, Schorderet D, et al. Nuclear receptor Rev-erb alpha (Nr1d1) functions in concert with Nr2e3 to regulate transcriptional networks in the retina. PLoS ONE. 2011;6:e17494. Benchorin G, Calton MA, Beaulieu MO, Vollrath D. Assessment of murine retinal function by electroretinography. Bio-Protoc. 2017;7:e2218. Haider NB, Zhang W, Hurd R, Ikeda A, Nystuen AM, Naggert JK, et al. Mapping of genetic modifiers of Nr2e3 rd7/rd7 that suppress retinal degeneration and restore blue cone cells to normal quantity. Mamm Genome Off J Int Mamm Genome Soc. 2008;19:145–54. Olivares AM, Jelcick AS, Reinecke J, Leehy B, Haider A, Morrison MA, et al. Multimodal regulation orchestrates normal and complex disease states in the retina. Sci Rep. 2017;7:690. Lotery AJ, Yang GS, Mullins RF, Russell SR, Schmidt M, Stone EM, et al. Adeno-associated virus type 5: transduction efficiency and cell-type specificity in the primate retina. Hum Gene Ther. 2003;14:1663–71. Yang GS, Schmidt M, Yan Z, Lindbloom JD, Harding TC, Donahue BA, et al. Virus-mediated transduction of murine retina with adeno-associated virus: effects of viral capsid and genome size. J Virol. 2002;76:7651–60. Pang JJ, Lauramore A, Deng WT, Li Q, Doyle TJ, Chiodo V, et al. Comparative analysis of in vivo and in vitro AAV vector transduction in the neonatal mouse retina: effects of serotype and site of administration. Vision Res. 2008;48:377–85. Frederick A, Sullivan J, Liu L, Adamowicz M, Lukason M, Raymer J, et al. Engineered capsids for efficient gene delivery to the retina and cornea. Hum Gene Ther. 2020;31:756–74. Kong F, Li W, Li X, Zheng Q, Dai X, Zhou X, et al. Self-complementary AAV5 vector facilitates quicker transgene expression in photoreceptor and retinal pigment epithelial cells of normal mouse. Exp Eye Res. 2010;90:546–54. Petit L, Ma S, Cheng SY, Gao G, Punzo C. Rod outer segment development influences AAV-mediated photoreceptor transduction after subretinal injection. Hum Gene Ther. 2017;28:464–81. Long BR, Sandza K, Holcomb J, Crockett L, Hayes GM, Arens J, et al. The impact of pre-existing immunity on the non-clinical pharmacodynamics of AAV5-based gene therapy. Mol Ther Methods Clin Dev. 2019;13:440–52. Hetz C, Glimcher LH. Fine tuning of the unfolded protein response: assembling the IRE1α interactome. Mol Cell. 2009;35:551–61. Alavi MV, Chiang WC, Kroeger H, Yasumura D, Matthes MT, Iwawaki T, et al. In vivo visualization of endoplasmic reticulum stress in the retina using the ERAI reporter mouse. Invest Ophthalmol Vis Sci. 2015;56:6961–70. Chiang WC, Messah C, Lin JH. IRE1 directs proteasomal and lysosomal degradation of misfolded rhodopsin. Mol Biol Cell. 2012;23:758–70.

Sujun Li , Shyamtanu Datta , Emily Brabbit , Zoe Love , Victoria Woytowicz , Kyle Flattery , Jessica Capri , Katie Yao , Siqi Wu , Michael Imboden , Arun Upadhyay , Rasappa Arumugham , Wallace B. Thoreson , Margaret M. DeAngelis , Neena B. Haider | Gene Therapy | 02 March 2020 | Vol 28 | pgs. 223–241 | doi.org/10.1038/s41434-020-0134-z Abstract Recent advances in viral vector engineering, as well as an increased understanding of the cellular and molecular mechanism of retinal diseases, have led to the development of novel gene therapy approaches. Furthermore, ease of accessibility and ocular immune privilege makes the retina an ideal target for gene therapies. In this study, the nuclear hormone receptor gene Nr2e3 was evaluated for efficacy as broad-spectrum therapy to attenuate early to intermediate stages of retinal degeneration in five unique mouse models of retinitis pigmentosa (RP). RP is a group of heterogenic inherited retinal diseases associated with over 150 gene mutations, affecting over 1.5 million individuals worldwide. RP varies in age of onset, severity, and rate of progression. In addition, ~40% of RP patients cannot be genetically diagnosed, confounding the ability to develop personalized RP therapies. Remarkably, Nr2e3 administered therapy resulted in reduced retinal degeneration as observed by increase in photoreceptor cells, improved electroretinogram, and a dramatic molecular reset of key transcription factors and associated gene networks. These therapeutic effects improved retinal homeostasis in diseased tissue. Results of this study provide evidence that Nr2e3 can serve as a broad-spectrum therapy to treat multiple forms of RP. Introduction Recent studies have demonstrated the potential of gene therapy to attenuate or slow the progression of previously untreatable inherited diseases [ 1 ]. Gene therapy has evolved from concept to clinic for various genetic disorders including hematological [ 2 ], immunological [ 3 ], ocular [ 4 , 5 ], neurodegenerative [ 6 ], and metabolic disorders [ 7 ]. Due to its ease of accessibility and immune privilege, the eye holds potential as an ideal organ for gene therapy. The most widely accepted success of adeno-associated virus (AAV) vector-mediated ocular gene replacement is for treatment of Leber’s congenital amaurosis 2 (LCA2), a rare retinal disease due to mutations in the RPE65 gene [ 8 , 9 ]. Currently, there are several ongoing clinical gene augmentation trials for other rare inherited retinal diseases [ 10 , 11 , 12 , 13 ]. However, several factors such as gene size, gene function, and the large number (~40%) of retinitis pigmentosa (RP) patients that cannot be genetically diagnosed present challenges for developing individual gene replacement/augmentation-based therapies. Thus, new therapeutic approaches are needed to circumvent these limitations. This study evaluates a unique approach using the nuclear hormone receptor (NHR) gene Nr2e3 as a genetic modifier and therapeutic agent to treat multiple retinal degenerative diseases. Results of this study demonstrate the power of a single genetic modifier in treating retinal diseases. RP represents a group of inherited diseases, affecting an estimated 1 in 4000 individuals, that cause degeneration of rod and cone photoreceptor cells, leading to the severe vision loss [ 14 , 15 ]. RP can be inherited through multiple modes of inheritance such as autosomal dominant (30–40% of cases), autosomal recessive (50–60% of cases), or X-linked (5–15% of cases) manner in syndromic or nonsyndromic forms [ 16 , 17 , 18 ]. Over 150 unique gene mutations have been associated with RP, making it highly heterogenic, with high variability in disease onset, severity, and progression [ 19 , 20 , 21 , 22 , 23 ]. Genetic modifiers are defined as allelic variants found within the normal population [ 24 , 25 ]. Modifier genes can significantly affect disease outcomes, impacting onset, rate of progression, and severity [ 24 , 26 , 27 ]. Genetic modifier genes are powerful modulators that can enhance or suppress disease phenotypes [ 24 , 27 , 28 , 29 ]. The direct impact of genetic modifiers has been studied extensively in several diseases including cystic fibrosis, epileptic encephalopathy, spinocerebellar ataxia type 1, spinal muscular atrophy, dystonia, and retinal degeneration where drastically altered phenotypes occur when genetic background is shifted [ 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ]. Haider et al. discovered that shifting the rd7 mutation, a recessive mutation in NHR 2 family e, member 3, Nr2e3 that results in slow progressive retinal degeneration, onto three different genetic backgrounds resulted in complete suppression of the rd7 phenotype in all strains evaluated, and genetic mapping revealed that several modifier genes could independently account for this suppression [ 39 ]. The NHR 1 family d, member 1 ( Nr1d1) , a NHR gene, and cofactor of Nr2e3 , was identified as one of the genetic modifiers that can ameliorate Nr2e3 associated retinal degeneration [ 40 ]. Mutations in human NR2E3 are associated with several forms of retinal degeneration that vary in phenotype and were categorized by their clinical diagnosis as they were discovered. These clinical categories include the recessive diseases enhanced S-cone syndrome (ESCS), Goldmann-Favre syndrome (GFS), and clumped pigmentary retinal degeneration (CPRD) [ 41 , 42 , 43 ]. NR2E3 mutations are also associated with up to 1% of all autosomal dominant retinitis pigmentosa (adRP) [ 44 , 45 ]. The association of NR2E3 with several clinical phenotypes and varying modes of inheritance strongly indicates that these retinal diseases manifest on a permissive or selective genetic background and are influenced, at least in part, by genetic modifier genes [ 46 , 47 , 48 , 49 ]. Given the role of NHRs such as NR2E3 , to modulate numerous key biological networks essential for maintaining retinal homeostasis, this study evaluated Nr2e3 as a broad-spectrum genetic modifier with the potential to attenuate retinal degeneration in several different mouse models. In this study, the efficacy of subretinal delivery of AAV8- Nr2e3 to attenuate and ameliorate retinal degeneration was assessed in five independent RP models that represent the heterogeneity observed in human RP disease. The five RP models tested were FVB- Pde6ß rd1 /NJ ( rd1 ), Rhodopsin null allele ( Rho −/−), B6.129S6(Cg)- Rhotm1.1Kpal /J ( RhoP23H ), BXD24/TyJ- Cep290rd16 /J ( rd16 ) and Nr2e3rd7 /J ( rd7 ) (Table  1 ). The rd1 mouse, representing the most severe and early form of human retinal degeneration, harbors a mutant Pde6b gene mapped on chromosome 5 [ 50 , 51 , 52 , 53 , 54 , 55 ]. The mutant Pde6b gene contains a murine leukemia provirus insertion in intron 1 and a point mutation, which introduces a stop codon in exon 7 (Y347STOP) [ 56 , 57 ]. Independent of this, a second mutation has been found in this gene, which is the integration of a murine leukemia virus in the first intron of the 6beta ( Pde6ß ) gene [ 58 ]. Mutations in human PDE6ß are associated with RP and autosomal dominant congenital stationary night blindness in humans [ 59 , 60 , 61 ]. The rhodopsin null ( Rho −/−) and the dominant negative RhoP23H alleles both lack a functional rhodopsin gene [ 62 , 63 ]. Rho −/− mice lack expression of rhodopsin mRNA and protein [ 64 ]. In contrast, RhoP23H mice are functional nulls with an amino acid substitution of proline to histidine at position 23 that generate an aberrant message leading to protein misfolding and degradation [ 63 ]. Specifically, RhoP23H protein undergoes incomplete glycosylation and is retained in the endoplasmic reticulum (ER) and/or Golgi apparatus where it is degraded [ 63 ]. Mutations in the human rhodopsin gene account for the largest portion of inherited retinal degenerations of known genetic etiology [ 65 ]. Further, the RhoP23H mutation in particular is one of the most commonly known causes of adRP in humans [ 63 ]. The Cep290rd16 ( rd16 ) mouse harbors a mutation in the centrosomal protein Cep290 that results in early-onset retinal degeneration with autosomal recessive inheritance [ 66 ]. Mutations in human CEP290 are associated with several syndromic and nonsyndromic forms of retinal degeneration [ 66 , 67 ]. The rd7 mouse is a model for Nr2e3 associated retinal degenerations. rd7 mice, harboring a recessive mutation in Nr2e3 , are clinically characterized by pan retinal spots apparent at eye opening (postnatal (P) day 14), and whorls and rosettes in the outer nuclear layer (ONL) observed histologically by P10 [ 68 , 69 ]. rd7 mice have two distinct outcomes: a disruption in development of cone cells causing a significant increase of blue opsin expressing cone cells, and progressive degeneration of rod and cone photoreceptor cells [ 68 , 70 ]. Results of this study show that the administration of AAV8- Nr2e3 therapy improves clinical, histological, functional, and molecular disease outcomes in each of the five models of retinal disease. These studies demonstrate the mechanism of Nr2e3 therapy involves resetting key retinal transcription factors and key biological networks that work in concert with Nr2e3 to modulate the homeostatic state of the retina. This research is predicated on the fact that disease outcome is rarely due to a single gene mutation; rather, it is a result of the combinatorial mutational load on the biological system, which is often strongly influenced by other factors such as modifier genes. This study demonstrates a novel approach to gene therapy and suggests that Nr2e3 can potentially serve as a broad-spectrum gene therapy to attenuate retinal degeneration. Click here to read entire paper References Russell S, Bennett J, Wellman JA, Chung DC, Yu ZF, Tillman A, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet. 2017;390:849–60. Braun CJ, Boztug K, Paruzynski A, Witzel M, Schwarzer A, Rothe M, et al. Gene therapy for Wiskott-Aldrich syndrome-long-term efficacy and genotoxicity. Sci Transl Med. 2014;7:357–65. Hacein-Bey-Abina S, Pai S-Y, Gaspar HB, Armant M, Berry CC, Blanche S, et al. A modified γ-retrovirus vector for X-linked severe combined immunodeficiency. N Engl J Med. 2014;371:1407–17. Bainbridge JWB, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K, et al. Effect of gene therapy on visual function in Leber’s congenital amaurosis. N Engl J Med. 2008;358:2231–9. Weleber RG, Pennesi ME, Wilson DJ, Kaushal S, Erker LR, Jensen L, et al. Results at 2 years after gene therapy for RPE65-deficient leber congenital amaurosis and severe early-childhood-onset retinal dystrophy. Ophthalmology. 2016;123:1606–20. Palfi S, Gurruchaga JM, Scott Ralph G, Lepetit H, Lavisse S, Buttery PC, et al. Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson’s disease: A dose escalation, open-label, phase 1/2 trial. Lancet. 2014;383:1138–46. Hwu WL, Muramatsu SI, Tseng SH, Tzen KY, Lee NC, Chien YH, et al. Gene therapy for aromatic L-amino acid decarboxylase deficiency. Sci Transl Med. 2012;4:134ra61. Marlhens F, Bareil C, Griffoin JM, Zrenner E, Amalric P, Eliaou C, et al. Mutations in RPE65 cause leber’s congenital amaurosis. Nat Genet. 1997;17:139–41. Acland GM, Aguirre GD, Ray J, Zhang Q, Aleman TS, Cideciyan AV, et al. Gene therapy restores vision in a canine model of childhood blindness. Nat Genet. 2001;12:1072–82. MacLaren RE, Groppe M, Barnard AR, Cottriall CL, Tolmachova T, Seymour L, et al. Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial. Lancet. 2014;383:1129–37. Lenis TL, Sarfare S, Jiang Z, Lloyd MB, Bok D, Radu RA. Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease. Proc Natl Acad Sci. 2017;114:3987–92. Ghazi NG, Abboud EB, Nowilaty SR, Alkuraya H, Alhommadi A, Cai H, et al. Treatment of retinitis pigmentosa due to MERTK mutations by ocular subretinal injection of adeno-associated virus gene vector: results of a phase I trial. Hum Genet. 2016;135:327–43. Kahle NA, Peters T, Zobor D, Kuehlewein L, Kohl S, Zhour A, et al. Development of methodology and study protocol: safety and efficacy of a single subretinal injection of rAAV.hCNGA3 in patients with CNGA3—linked achromatopsia investigated in an exploratory dose-escalation trial. Hum Gene Ther Clin Dev. 2018;29:121–31. Parmeggiani F, Sorrentino FS, Ponzin D, Barbaro V, Ferrari S, Di Iorio E. Retinitis Pigmentosa: Genes and disease mechanisms. Curr Genom. 2011;12:238–49. Daiger SP, Sullivan LS, Bowne SJ. Genes and mutations causing retinitis pigmentosa. Clin Genet. 2013;84:132– Bunker C, Berson E, Bromley W, Hayes R, Roderick T. Prevalence of retinitis pigmentosa in Maine. Am J Ophthalmol. 1984;97:357–65. Grøndahl J. Estimation of prognosis and prevalence of retinitis pigmentosa and Usher syndrome in Norway. Clin Genet. 1987;31:255–64. Pierrottet CO, Zuntini M, Digiuni M, Bazzanella I, Ferri P, Paderni R, et al. Syndromic and non-syndromic forms of retinitis pigmentosa: a comprehensive Italian clinical and molecular study reveals new mutations. Genet Mol Res. 2014;13:8815–33. Ali MU, Rahman MSU, Cao J, Yuan PX. Genetic characterization and disease mechanism of retinitis pigmentosa; current scenario. 3 Biotech. 2017;7:251. Al-maghtheh M, Inglehearn CF, Jeffrey TK, Evans K, Moore AT, Jay M, et al. Identification of a sixth locus for autosomal dominant retinitis pigmentosa on chromosome 19. Hum Mol Genet. 1994;3:351–4. Article   CAS   PubMed   Google Scholar   Andréasson S, Ponjavic V, Abrahamson M, Ehinger B, Wu W, Fujita R, et al. Phenotypes in three Swedish families with X-linked retinitis pigmentosa caused by different mutations in the RPGR gene. Am J Ophthalmol. 1997;124:95–102. Article   PubMed   Google Scholar   Blanton SH, Heckenlively JR, Cottingham AW, Friedman J, Sadler LA, Wagner M, et al. Linkage mapping of autosomal dominant retinitis pigmentosa (RP1) to the pericentric region of human chromosome 8. Genomics. 1991;11:857–69. Article   CAS   PubMed   Google Scholar   Hamel C, Hartong DT, Berson EL, Dryja TP, Hamel C. Retinitis pigmentosa. Orphanet J Rare Dis. 2006;1:40. Article   PubMed   PubMed Central   Google Scholar   Houlston RS, Tomlinson IP. Modifier genes in humans: strategies for identification. Eur J Hum Genet. 1998;6:80–8. Article   CAS   PubMed   Google Scholar   Harper AR, Nayee S, Topol EJ. Protective alleles and modifier variants in human health and disease. Nat Rev Genet. 2015;16:689–701. Article   CAS   PubMed   Google Scholar   Chow CY, Kelsey KJP, Wolfner MF, Clark AG. Candidate genetic modifiers of retinitis pigmentosa identified by exploiting natural variation in Drosophila . Hum Mol Genet. 2016;25:651–9. Article   CAS   PubMed   Google Scholar   Haider NB, Ikeda A, Naggert JK, Nishina PM. Genetic modifiers of vision and hearing. Hum Mol Genet. 2002;11:1195–206. Article   CAS   PubMed   Google Scholar   Salvatore F, Scudiero O, Castaldo G. Genotype-phenotype correlation in cystic fibrosis: the role of modifier genes. Am J Med Genet. 2002;111:88–95. Article   PubMed   Google Scholar   Dipple KM, McCabe ERB. Modifier genes convert “simple” Mendelian disorders to complex traits. Mol Genet Metab. 2000;71:43–50. Article   CAS   PubMed   Google Scholar   Hsieh CS, Macatonia SE, Garra A, Murphy KM. T cell genetic background determines default T helper phenotype development in vitro. J Exp Med. 1995;181:713 LP–721. Article   Google Scholar   Kiesewetter S, Macek M, Davis C, Curristin SM, Chu CS, Graham C, et al. A mutation in CFTR produces different phenotypes depending on chromosomal background. Nat Genet. 1993;5:274–8. Article   CAS   PubMed   Google Scholar   Rose-Hellekant TA, Gilchrist K, Sandgren EP. Strain background alters mammary gland lesion phenotype in transforming growth factor-α transgenic mice. Am J Pathol. 2002;161:1439–47. Article   CAS   PubMed   PubMed Central   Google Scholar   Calhoun JD, Hawkins NA, Zachwieja NJ, Kearney JA. Cacna1g is a genetic modifier of epilepsy in a mouse model of Dravet syndrome. Epilepsia. 2017;58:e111–5. Eshraghi M, McFall E, Gibeault S, Kothary R. Effect of genetic background on the phenotype of the Smn 2B/-mouse model of spinal muscular atrophy. Hum Mol Genet. 2016;25:ddw278. Tanabe LM, Martin C, Dauer WT. Genetic background modulates the phenotype of a mouse model of dyt1 dystonia. PLoS ONE. 2012;7:e32245. Ebermann I, Phillips JB, Liebau MC, Koenekoop RK, Schermer B, Lopez I, et al. PDZD7 is a modifier of retinal disease and a contributor to digenic Usher syndrome. J Clin Investig. 2010;120:1812–23. Maddox DM, Ikeda S, Ikeda A, Zhang W, Krebs MP, Nishina PM, et al. An allele of microtubule-associated protein 1A (Mtap1a) reduces photoreceptor degeneration in Tulp1 and tub mutant mice. Investig Ophthalmol Vis Sci. 2012;53:1663–9. Fernandez-Funez P, Nino-Rosales ML, De Gouyon B, She WC, Luchak JM, Martinez P, et al. Identification of genes that modify ataxin-1-induced neurodegeneration. Nature. 2000;408:101–6. Haider NB, Zhang W, Hurd R, Ikeda A, Nystuen AM, Naggert JK, et al. Mapping of genetic modifiers of Nr2e3 rd7/rd7 that suppress retinal degeneration and restore blue cone cells to normal quantity. Mamm Genome. 2008;19:145–54. Article   CAS   PubMed   Google Scholar   Cruz NM, Yuan Y, Leehy BD, Baid R, Kompella U, DeAngelis MM, et al. Modifier genes as therapeutics: the nuclear hormone receptor rev erb alpha (Nr1d1) rescues Nr2e3 associated retinal disease. PLoS ONE. 2014;9:e87942. Haider N, Jacobson S, Cideciyan A, Swiderski R, Streb L, Searby C, et al. Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate. Nat Genet. 2000;24:127–31. Article   CAS   PubMed   Google Scholar   Schorderet DF, Escher P. NR2E3 mutations in enhanced S-cone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), clumped pigmentary retinal degeneration (CPRD), and retinitis pigmentosa (RP). Hum Mutat. 2009;30:1475–85. Article   CAS   PubMed   Google Scholar   Sharon D, Sandberg MA, Caruso RC, Berson EL, Dryja TP. Shared mutations in NR2E3 in enhanced S-cone syndrome, Goldmann-Favre syndrome, and many cases of clumped pigmentary retinal degeneration. Arch Ophthalmol. 2003;121:1316–23. Article   CAS   PubMed   Google Scholar   Coppieters F, Leroy BP, Beysen D, Hellemans J, De Bosscher K, Haegeman G, et al. Recurrent mutation in the first zinc finger of the orphan nuclear receptor NR2E3 causes autosomal dominant retinitis pigmentosa. Am J Hum Genet. 2007;81:147–57. Article   CAS   PubMed   PubMed Central   Google Scholar   Gire A, Sullivan L, Bowne S, Birch D, Hughbanks-Wheaton D, Heckenlively JR, et al. The Gly56Arg mutation in NR2E3 accounts for 1-2% of autosomal dominant retinitis pigmentosa. Mol Vis. 2007;13:1970–5. CAS   PubMed   Google Scholar   Webber AL, Hodor P, Thut CJ, Vogt TF, Zhang T, Holder DJ, et al. Dual role of Nr2e3 in photoreceptor development and maintenance. Exp Eye Res. 2008;87:35–48. Article   CAS   PubMed   Google Scholar   Cheng H, Aleman TS, Cideciyan AV, Khanna R, Jacobson SG, Swaroop A. In vivo function of the orphan nuclear receptor NR2E3 in establishing photoreceptor identity during mammalian retinal development. Hum Mol Genet. 2006;15:2588–602. Article   CAS   PubMed   Google Scholar   Haider NB, Demarco P, Nystuen AM, Huang X, Smith RS, Mccall MA, et al. The transcription factor Nr2e3 functions in retinal progenitors to suppress cone cell generation. Vis Neurosci. 2006;23:917–29. Article   PubMed   Google Scholar   Olivares AM, Jelcick AS, Reinecke J, Leehy B, Haider A, Morrison MA, et al. Multimodal regulation orchestrates normal and complex disease states in the retina. Sci Rep. 2017;7:690–706. Article   CAS   PubMed   PubMed Central   Google Scholar   Sidman RL, Green MC. Retinal degeneration in the mouse: location of the rd locus in linkage group xvii. J Hered. 1965;56:23–9. Lolley RN, Farber DB, Rayborn ME, Hollyfield JG. Cyclic gmp accumulation causes degeneration of photoreceptor cells: simulation of an inherited disease. Science (80-). 1977. https://doi.org/10.1126/science.193183 . Farber DB. From mice to men: the cyclic GMP phosphodiesterase gene in vision and disease: the proctor lecture. Investig Ophthalmol Vis Sci. 1995;36:263–75. Jones BW, et al. Retinal remodeling triggered by photoreceptor degenerations. J Comp Neurol. 2003. https://doi.org/10.1002/cne.10703 . Peng YW, Hao Y, Petters RM, Wong F. Ectopic synaptogenesis in the mammalian retina caused by rod photoreceptor-specific mutations. Nat Neurosci. 2000. https://doi.org/10.1038/80639 . Strettoi E, Porciatti V, Falsini B, Pignatelli V, Rossi C. Morphological and functional abnormalities in the inner retina of the rd/rd mouse. J. Neurosci. 2002. https://doi.org/10.1523/jneurosci.22-13-05492.2002 . Bowes C, Li T, Frankel WN, Danciger M, Coffin JM, Applebury ML, et al. Localization of a retroviral element within the rd gene coding for the β subunit of cGMP phosphodiesterase. Proc Natl Acad Sci USA. 1993;90:2955–9. Pittler SJ, Baehr W. Identification of a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase β-subunit gene of the rd mouse. Proc Natl Acad Sci USA. 1991;88:8322–6 Bowes C, Li T, Danciger M, Baxter LC, Applebury ML, Farber DB. Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase. Nature. 1990;347:677–80. Article   CAS   PubMed   Google Scholar   Danciger M, Blaney J, Gao Yq, Zhao Dy, Heckenlively Jr, Jacobson SG, et al. Mutations in the PDE6B gene in autosomal recessive retinitis pigmentosa. Genomics. 1995;30:1–7. Article   CAS   PubMed   Google Scholar   Gopalakrishna KN, Boyd K, Artemyev NO. Mechanisms of mutant PDE6 proteins underlying retinal diseases. Cell Signal. 2017;37:74–80. Article   CAS   PubMed   PubMed Central   Google Scholar   McLaughlin ME, Ehrhart TL, Berson EL, Dryja TP. Mutation spectrum of the gene encoding the beta subunit of rod phosphodiesterase among patients with autosomal recessive retinitis pigmentosa. Proc Natl Acad Sci USA. 1995;92:3249–53. Article   CAS   PubMed   PubMed Central   Google Scholar   Humphries MM, Rancourt D, Farrar GJ, Kenna P, Hazel M, Bush RA, et al. Retinopathy induced in mice by targeted disruption of the rhodopsin gene. Nat Genet. 1997;15:216–9. Article   CAS   PubMed   Google Scholar   Sakami S, Maeda T, Bereta G, Okano K, Golczak M, Sumaroka A, et al. Probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to P23H opsin mutations. J Biol Chem. 2011;286:10551–67. Article   CAS   PubMed   PubMed Central   Google Scholar   Lem J, Krasnoperova NV, Calvert PD, Kosaras B, Cameron DA, Nicolò M, et al. Morphological, physiological, and biochemical changes in rhodopsin knockout mice. Proc Natl Acad Sci USA. 1999;96:736–41. Article   CAS   PubMed   PubMed Central   Google Scholar   Shokravi MT, Dryja TP. Retinitis pigmentosa and the rhodopsin gene. Int Ophthalmol Clin. 1993;33:219–28. Chang B, Khanna H, Hawes N, Jimeno D, He S, Lillo C, et al. In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse. Hum Mol Genet. 2006;15:1847–57. Article   CAS   PubMed   Google Scholar   Shen T, Guan L, Li S, Zhang J, Xiao X, Jiang H, et al. Mutation analysis of Leber congenital amaurosis-associated genes in patients with retinitis pigmentosa. Mol Med Rep. 2015;11:1827–32. Article   CAS   PubMed   Google Scholar   Akhmedov NB, Piriev NI, Chang B, Rapoport AL, Hawes NL, Nishina PM, et al. A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse. Proc Natl Acad Sci USA. 2000;97:5551–6. Article   CAS   PubMed   PubMed Central   Google Scholar   Haider N, Naggert J, Nishina P. Excess cone cell proliferation due to lack of a functional NR2E3 causes retinal dysplasia and degeneration in rd7/rd7 mice. Hum Mol Genet. 2001;10:1619–26. Article   CAS   PubMed   Google Scholar   Haider NB, Naggert JK, Nishina PM. Excess cone cell proliferation due to lack of a functional NR2E3 causes retinal dysplasia and degeneration in rd7/rd7 mice. Hum Mol Genet. 2001;10:1619–26. Article   CAS   PubMed   Google Scholar   Peng G, Ahmad O, Ahmad F, Liu J, Chen S. The photoreceptor-specific nuclear receptor Nr2e3 interacts with Crx and exerts opposing effects on the transcription of rod versus cone genes. Human molecular genetics. Hum Mol Genet. 2005;14:747–64. Article   CAS   PubMed   Google Scholar

Zhi-Hong Zhu , Xin Jin , Yi-Xin Zhang , Rui Wang , Tong Wu , Wei Liu , Ze-Hua Chen , Hai-Nan Xie , Lan-Lan Chen , Zi-Hao Liu , Hou-Bin Huang | International Journal Ophthalmology | 2022 Feb 18 | 15(2) | pages 205–212 | doi: 10.18240/ijo.2022.02.03 AIM To describe the clinical heterogeneity of patients with novel mutations in BEST1 . METHODS All the members in the two Chinese families underwent detailed clinical evaluations including best-corrected visual acuity, slit-lamp examination, applanation tonometry, and dilated fundus examination. Fundus autofluorescence, fundus fluorescein angiography, spectral-domain optical coherence tomography, electrooculography, and electroretinogram were also performed. Genomic DNA was extracted from venous blood for all the participants. The targeted next-generation sequencing of inherited retinal disease-associated genes was conducted to identify the causative mutation. RESULTS A novel BEST1 missense mutation c.41T>C (p.Leu14Ser) was identified in Family 1. It was co-segregated with the phenotype of best vitelliform macular dystrophy (BVMD) and bioinformatics analysis confirmed it was harmful. Another novel BEST1 frameshift mutation c.345_346insGGCAAGGACG (p.Glu119Glyfs*116) and a novel USH2A missense mutation c.12560G>A, p.Arg4187His were identified in family 2 with retinitis pigmentosa (RP), which might interact and lead to the phenotype of RP. CONCLUSION Two novel mutations in the BEST1 gene in two unrelated families with distinct phenotypes and BEST1 mutation accompanied with USH2A mutation would result in RP, which could be enormously helpful in understanding the pathogenesis of the inherited retinal disease caused by a BEST1 mutation. Click here to read the entire article Reference Johnson AA, Guziewicz KE, Lee CJ, Kalathur RC, Pulido JS, Marmorstein LY, Marmorstein AD. Bestrophin 1 and retinal disease. Prog Retin Eye Res 2017;58:45-69.<br> Lin Y, Li T, Gao HB, Lian Y, Chen C, Zhu Y, Li YH, Liu BQ, Zhou WL, Jiang HY, Liu XL, Zhao XJ, Liang XL, Jin CJ, Huang XH, Lu L. Bestrophin 1 gene analysis and associated clinical findings in a Chinese patient with Best vitelliform macular dystrophy. Mol Med Rep 2017;16(4):4751-4755.<br> Smith JJ, Nommiste B, Carr AJF. Bestrophin1: a gene that causes many diseases. Adv Exp Med Biol 2019;1185:419-423.<br> Marmorstein AD, Johnson AA, Bachman LA, Andrews-Pfannkoch C, Knudsen T, Gilles BJ, Hill M, Gandhi JK, Marmorstein LY, Pulido JS. Mutant BEST1 expression and impaired phagocytosis in an iPSC model of autosomal recessive bestrophinopathy. Sci Rep 2018;8:4487.<br> Marmorstein AD, Kinnick TR, Stanton JB, Johnson AA, Lynch RM, Marmorstein LY. Bestrophin-1 influences transepithelial electrical properties and Ca2+ signaling in human retinal pigment epithelium. Mol Vis 2015;21:347-359.<br> Gao TT, Tian CQ, Xu H, Tang X, Huang LZ, Zhao MW. Disease-causing mutations associated with bestrophinopathies promote apoptosis in retinal pigment epithelium cells. Graefes Arch Clin Exp Ophthalmol 2020;258(10):2251-2261.<br> Katagiri S, Hayashi T, Ohkuma Y, Sekiryu T, Takeuchi T, Gekka T, Kondo M, Iwata T, Tsuneoka H. Mutation analysis of BEST1 in Japanese patients with Best's vitelliform macular dystrophy. Br J Ophthalmol 2015;99(11):1577-1582.<br> Guziewicz KE, Sinha D, Gómez NM, Zorych K, Dutrow EV, Dhingra A, Mullins RF, Stone EM, Gamm DM, Boesze-Battaglia K, Aguirre GD. Bestrophinopathy: an RPE-photoreceptor interface disease. Prog Retin Eye Res 2017;58:70-88.<br> Fister TA, Zein WM, Cukras CA, Sen HN, Maldonado RS, Huryn LA, Hufnagel RB. Phenotypic and genetic spectrum of autosomal recessive bestrophinopathy and best vitelliform macular dystrophy. Invest Ophthalmol Vis Sci 2021;62(6):22.<br> Best F. Über eine hereditäre Maculaaffektion. Ophthalmologica. Journal international d'ophtalmologie. International journal of ophthalmology. Zeitschrift fur Augenheilkunde 1905;13(3):199-212.<br> Lin Y, Li T, Ma CH, Gao HB, Chen C, Zhu Y, Liu BQ, Lian Y, Huang Y, Li HC, Wu QX, Liang XL, Jin CJ, Huang XH, Ye JH, Lu L. Genetic variations in Bestrophin1 and associated clinical findings in two Chinese patients with juvenileonset and adultonset best vitelliform macular dystrophy. Mol Med Rep 2018;17(1):225-233.<br> sang SH, Sharma T. Best vitelliform macular dystrophy. Adv Exp Med Biol 2018;1085:79-90.<br> Parodi MB, Arrigo A, Bandello F. Reply: natural course of the vitelliform stage in best vitelliform macular dystrophy: a five-year follow-up study. Graefes Arch Clin Exp Ophthalmol 2021;259(3):789-790.<br> Burgess R, Millar ID, Leroy BP, Urquhart JE, Fearon IM, De Baere E, Brown PD, Robson AG, Wright GA, Kestelyn P, Holder GE, Webster AR, Manson FD, Black GC. Biallelic mutation of BEST1 causes a distinct retinopathy in humans. Am J Hum Genet 2008;82(1):19-31.<br> Kaufman SJ, Goldberg MF, Orth DH, Fishman GA, Tessler H, Mizuno K. Autosomal dominant vitreoretinochoroidopathy. Arch Ophthalmol 1982;100(2):272-278.<br> Davidson AE, Millar ID, Urquhart JE, Burgess-Mullan R, Shweikh Y, Parry N, O'Sullivan J, Maher GJ, McKibbin M, Downes SM, Lotery AJ, Jacobson SG, Brown PD, Black GCM, Manson FDC. Missense mutations in a retinal pigment epithelium protein, bestrophin-1, cause retinitis pigmentosa. Am J Hum Genet 2009;85(5):581-592.<br> Dalvin LA, Abou Chehade JE, Chiang J, Fuchs J, Iezzi R, Marmorstein AD. Retinitis pigmentosa associated with a mutation in BEST1. Am J Ophthalmol Case Rep 2016;2:11-17.<br> Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 2010;26(5):589-595.<br> Wang K, Li MY, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 2010;38(16):e164.<br> Auton A, Abecasis GR, Altshuler DM, et al. A global reference for human genetic variation. Nature 2015;526(7571):68-74.<br> Jin X, Chen LL, Wang DJ, Zhang YX, Chen ZH, Huang HB. Novel compound heterozygous mutation in the POC1B gene underlie peripheral cone dystrophy in a Chinese family. Ophthalmic Genet 2018;39(3):300-306.<br> Jin X, Liu W, Qv LH, Huang HB. A novel variant in PAX6 as the cause of aniridia in a Chinese family. BMC Ophthalmol 2021;21(1):225.<br> Jin X, Qu LH, Hou BK, Xu HW, Meng XH, Pang CP, Yin ZQ. Novel compound heterozygous mutation in the CNGA1 gene underlie autosomal recessive retinitis pigmentosa in a Chinese family. Biosci Rep 2016;36(1):e00289.<br> Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL, Committee ACMGLQA. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17(5):405-424.<br> Ji CY, Li Y, Kittredge A, Hopiavuori A, Ward N, Yao P, Fukuda Y, Zhang Y, Tsang SH, Yang TT. Investigation and restoration of BEST1 activity in patient-derived RPEs with dominant mutations. Sci Rep 2019;9(1):19026.<br> Owji AP, Kittredge A, Zhang Y, Yang TT. Structure and Function of the Bestrophin family of calcium-activated chloride channels. Channels (Austin) 2021;15(1):604-623.<br> Uggenti C, Briant K, Streit AK, Thomson S, Koay YH, Baines RA, Swanton E, Manson FD. Restoration of mutant bestrophin-1 expression, localisation and function in a polarised epithelial cell model. Dis Model Mech 2016;9(11):1317-1328.<br> Singh R, Kuai D, Guziewicz KE, Meyer J, Wilson M, Lu JF, Smith M, Clark E, Verhoeven A, Aguirre GD, Gamm DM. Pharmacological modulation of photoreceptor outer segment degradation in a human iPS cell model of inherited macular degeneration. Mol Ther 2015 23(11):1700-1711.<br> Gao TT, Tian CQ, Hu QR, Liu ZM, Zou JM, Huang LZ, Zhao MW. Clinical and mutation analysis of patients with best vitelliform macular dystrophy or autosomal recessive bestrophinopathy in Chinese population. Biomed Res Int 2018;2018:4582816.<br> Guziewicz KE, Cideciyan AV, Beltran WA, Komáromy AM, Dufour VL, Swider M, Iwabe S, Sumaroka A, Kendrick BT, Ruthel G, Chiodo VA, Héon E, Hauswirth WW, Jacobson SG, Aguirre GD. BEST1 gene therapy corrects a diffuse retina-wide microdetachment modulated by light exposure. Proc Natl Acad Sci U S A 2018;115(12): E2839-E2848.<br> Singh Grewal S, Smith JJ, Carr AJF. Bestrophinopathies: perspectives on clinical disease, Bestrophin-1 function and developing therapies. Ther Adv Ophthalmol 2021;13:2515841421997191.

Photoreceptor cells are susceptible to cellular stress - their degeneration and loss is a major cause of blindness. Many genes have identified for the inherited and highly heterogeneous disorders resulting and its patterns of inheritance are varied - some are autosomal dominant (adRP), others are autosomal recessive (arRP), a smaller fraction are X-linked (XLRP) and between 30 to 50% have not yet been classified. Other disorders include Bardet-Biedl syndrome (BBS), macular and age-related macular degeneration (MD, AMD), Leber congenital amaurosis (LCA), cone and cone-rod degeneration (CD, CRD). RP may occur alone or non-syndromic or in combination with other disorders, such as the Usher syndrome. Mutations in the same gene can cause different phenotypes, such as the many mutations in rhodopsin receptor (Rho) that cause adRP or arRP. Many RP mutations are in genes involved in the phototransduction and the metabolic visual cycle pathways. The response to light in the vertebrate retina is mediated by two photoreceptor types: the rods that mediate vision in dim light and the cones that mediate bright light and color vision. Both are G-protein coupled receptors (GPCR) that activate the specific heterotrimeric G protein transducin complex upon their own activation by the visual pigment - the vitamin A-derived 11-cis retinal. The one rod gene (Rho) and three cone genes are collectively known as opsins. Some 200 point mutations have been described for Rho and are associated with both adRP and arRP; they have been categorized into six classes with class I and II being the more common. Upon activation, transducin activates the cGMP phoshodiesterase (Pde) complex; the subsequent decrease in cGMP closes the cGMP-gated cation channels resulting in decreased calcium (Ca2+) influx. Mutations in rod-specific Pde and in cGMP-gated channels are associated with arRP. Decrease in intracellular Ca2+ also promotes the activation of guanylate cyclases and restoration of cGMP levels. The enzymes are constitutively bound to activator proteins (GCAPs), Ca2+ binding proteins that inhibit the cyclases in the presence of Ca2+ but stimulate them in its absence. Mutations in an activator gene have been associated with adRP. GPCR signaling is controlled by several classes of proteins - the kinases that phosphorylate the activated receptors which are then recognized by arrestins whose binding precludes re-binding of G-proteins; at the G-protein level, by GTPase-activating proteins (GAPs) that increase the rate of G-protein GTP hydrolysis leading to inactivation of the Galpha subunit. SAG is a specific arrestin whose mutations have been associated with arRP. In the absence of light, 11-cis retinal acts as an inverse agonist that constrains the receptor in an inactive conformation. Click here to read entire article and learn more about pathways. References Daiger SP, et al. | Clin Genet | 2013 Aug | 84(2) | 132-41 | doi: 10.1111/cge.12203 Wright AF, et al. | Nat Rev Genet | 2010 Apr | 11(4) | 273-84 | doi: 10.1038/nrg2717 Athanasiou D, et al. | FEBS Lett | 2013 Jun 27 | 587(13) | 2008-17 | doi: 10.1016/j.febslet.2013.05.020 Mendes HF, et al. | Trends Mol Med | 2005 Apr | 11(4) | 177-85 Jones BW, et al. | Jpn J Ophthalmoly | 2012 July | 56(4) | 289-306 | doi: 10.1007/s10384-012-0147-2 Petrs-Silva H and Linden R | Clin Ophthalmoly | 2014 | 8:127-136. Estrada-Cuzcano A, et al. | Hum Mol Genet | 2012 Oct 15 | 21(R1) | R111-24 Xu S | Prog Retin Eye Res | 2009 Mar | 28(2) | 87-116 | doi: 10.1016/j.preteyeres.2008.11.00 Liu MM, et al. | Curr Genomics | 2013 May | 14(3) | 166-72 | doi: 10.2174/1389202911314030002 Huang KM, et al. | Mamm Genome | 2008 Aug | 19(7-8) | 510-6 | doi: 10.1007/s00335-008-9127-8 Pan L and Zhang M | Physiology (Bethesda) | 2012 Feb | 27(1) | 25-42 | doi: 10.1152/physiol.00037.2011

Two agree to licensing, co-development, and commercialization agreement for Europe for CTx-PDE6b, a novel gene therapy candidate in retinitis pigmentosa Press Release Paris, France, 16th September 2021 – Coave Therapeutics (‘Coave’), a clinical-stage biotechnology company focused on developing life-changing gene therapies in rare Ocular and CNS (Central Nervous System) diseases, has entered into an exclusive licensing, co-development and commercialization agreement with Théa, a leading European speciality Pharma focused on ophthalmology. The agreement covers Coave’s lead investigational gene therapy candidate CTx-PDE6b (formerly HORA-PDE6b) for patients with PDE6b-associated Retinitis Pigmentosa (RP), an inherited retinal disease (IRD). “We are delighted to sign this agreement with Théa for CTx-PDE6b, our most advanced candidate, which represents an innovative new gene therapy for people with Retinitis Pigmentosa,” said Rodolphe Clerval, CEO of Coave . “Théa is a leading European speciality pharma company in the field of Ophthalmology with world-class development expertise and commercial capabilities in major markets worldwide. We are very excited about the potential of CTx-PDE6b and look forward to building a successful relationship with Théa, as we advance this novel candidate through clinical development.” To read entire press release click here Contact Information Coave Therapeutics Rodolphe Clerval, CEO contact@coavetx.com MEDiSTRAVA Consulting Sylvie Berrebi, Eleanor Perkin, Mark Swallow PhD coavetx@medistrava.com Tel: +44 (0)7714 306525 Laboratoires Théa Lorraine Kaltenbach, Director of Communication Lorraine.KALTENBACH@theapharma.com

Vasily Smirnov, Marco Nassisi, Saddek Mohand-Said, Crystel Bonnet, Anne Aubois, Céline Devisme, Thilissa Dib, Christina Zeitz, Natalie Loundon, Sandrine Marlin, Christine Petit, Bahram Bodaghi, José-Alain Sahel, Isabelle Audo| April 2022 | Investigative Ophthalmology & Visual Science | 63(4) | Pg. 25 | doi: 1 0.1167/iovs.63.4.25 Abstract Purpose : Biallelic variants in CLRN1 are responsible for Usher syndrome 3A and non-syndromic rod–cone dystrophy (RCD). Retinal findings in Usher syndrome 3A have not been well defined. We report the detailed phenotypic description of RCD associated with CLRN1 variants in a prospective cohort. Methods : Patients were clinically investigated at the National Reference Center for rare ocular diseases at the Quinze-Vingts Hospital, Paris, France. Best-corrected visual acuity (BCVA) tests, Goldmann perimetry, full-field electroretinography (ffERG), retinal photography, near-infrared reflectance, short-wavelength and near-infrared autofluorescence, and optical coherence tomography (OCT) were performed for all patients. Results : Four patients from four unrelated families were recruited. Mean follow-up was 11 years for three patients, and only baseline data were available for one subject. Median BCVA at baseline was 0.2 logMAR (range, 0.3–0). ffERG responses were undetectable in all subjects. The III4e isopter of the Goldmann visual field was constricted to 10°. The retinal phenotype was consistent in all patients: small whitish granular atrophic areas were organized in a network pattern around the macula and in the midperiphery. OCT showed intraretinal microcysts in all patients. Upon follow-up, all patients experienced a progressive BCVA loss and further visual field constriction. Four distinct pathogenic variants were identified in our patients: two missense (c.144T>G, p.(Asn48Lys) and c.368C>A, p.(Ala123Asp)) and two frameshift variants (c.176del, p.(Gly59Valfs*13) and c.230dup, p.(Ala78Serfs*52)). Conclusions : RCD in Usher 3A syndrome has some distinctive features. It is a severe photoreceptor dystrophy with whitish granular posterior pole appearance and cystic maculopathy. Click here for link to source document. Read entire paper. References Boughman JA, Vernon M, Shaver KA. Usher syndrome: definition and estimate of prevalence from two high-risk populations. J Chronic Dis . 1983; 36(8): 595–603. Hartong DT, Berson EL, Dryja TP. Retinitis pigmentosa. Lancet . 2006; 368(9549): 1795–1809. Keats BJ, Corey DP. The usher syndromes. Am J Med Genet . 1999; 89(3): 158–166. Kimberling WJ, Hildebrand MS, Shearer AE, et al. Frequency of Usher syndrome in two pediatric populations: implications for genetic screening of deaf and hard of hearing children. Genet Med . 2010; 12(8): 512–516. Mathur PD, Yang J. Usher syndrome and non-syndromic deafness: functions of different whirlin isoforms in the cochlea, vestibular organs, and retina. Hear Res . 2019; 375: 14–24. Heissler SM, Manstein DJ. Functional characterization of the human myosin-7a motor domain. Cell Mol Life Sci . 2012; 69(2): 299–311. Weil D, Blanchard S, Kaplan J, et al. Defective myosin VIIA gene responsible for Usher syndrome type 1B. Nature . 1995; 374(6517): 60–61. Bonnet C, El-Amraoui A. Usher syndrome (sensorineural deafness and retinitis pigmentosa): pathogenesis, molecular diagnosis and therapeutic approaches. Curr Opin Neurol . 2012; 25(1): 42–49. Bhattacharya G, Kalluri R, Orten DJ, Kimberling WJ, Cosgrove D. A domain-specific usherin/collagen IV interaction may be required for stable integration into the basement membrane superstructure. J Cell Sci . 2004; 117(2): 233–242. Eudy JD, Weston MD, Yao S, et al. Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa. Science . 1998; 280(5370): 1753–1757. Seyedahmadi BJ, Rivolta C, Keene JA, Berson EL, Dryja TP. Comprehensive screening of the USH2A gene in Usher syndrome type II and non-syndromic recessive retinitis pigmentosa. Exp Eye Res . 2004; 79(2): 167–173. Ávila-Fernández A, Cantalapiedra D, Aller E, et al. Mutation analysis of 272 Spanish families affected by autosomal recessive retinitis pigmentosa using a genotyping microarray. Mol Vis . 2010; 16: 2550–2558. McGee TL, Seyedahmadi BJ, Sweeney MO, Dryja TP, Berson EL. Novel mutations in the long isoform of the USH2A gene in patients with Usher syndrome type II or non-syndromic retinitis pigmentosa. J Med Genet . 2010; 47(7): 499–506. Nuutila A. Dystrophia retinae pigmentosa–dysacusis syndrome (DRD): a study of the Usher- or Hallgren syndrome. J Genet Hum . 1970; 18(1): 57–88. Gorlin RJ, Tilsner TJ, Feinstein S, Duvall AJ. Usher's syndrome type III. Arch Otolaryngol Chic Ill 1960 . 1979; 105(6): 353–354. Sadeghi M, Cohn ES, Kimberling WJ, Tranebjaerg L, Möller C. Audiological and vestibular features in affected subjects with USH3 : a genotype/phenotype correlation. Int J Audiol . 2005; 44(5): 307–316. Adato A, Vreugde S, Joensuu T, et al. USH3A transcripts encode clarin-1, a four-transmembrane-domain protein with a possible role in sensory synapses. Eur J Hum Genet . 2002; 10(6): 339–350. Abbott JA, Meyer-Schuman R, Lupo V, et al. Substrate interaction defects in histidyl-tRNA synthetase linked to dominant axonal peripheral neuropathy. Hum Mutat . 2018; 39(3): 415–432. Khan MI, Kersten FFJ, Azam M, et al. CLRN1 mutations cause nonsyndromic retinitis pigmentosa. Ophthalmology . 2011 Jul 1; 118(7): 1444–1448. Puffenberger EG, Jinks RN, Sougnez C, et al. Genetic mapping and exome sequencing identify variants associated with five novel diseases. PLoS One . 2012; 7(1): e28936. Vester A, Velez-Ruiz G, McLaughlin HM, et al. A loss-of-function variant in the human histidyl-tRNA synthetase ( HARS ) gene is neurotoxic in vivo. Hum Mutat . 2013; 34(1): 191–199. Safka Brozkova D, Deconinck T, Griffin LB, et al. Loss of function mutations in HARS cause a spectrum of inherited peripheral neuropathies. Brain J Neurol . 2015; 138(Pt 8): 2161–2172. Galatolo D, Kuo ME, Mullen P, et al. Bi-allelic mutations in HARS1 severely impair histidyl-tRNA synthetase expression and enzymatic activity causing a novel multisystem ataxic syndrome. Hum Mutat . 2020; 41(7): 1232–1237. Bonnet C, Riahi Z, Chantot-Bastaraud S, et al. An innovative strategy for the molecular diagnosis of Usher syndrome identifies causal biallelic mutations in 93% of European patients. Eur J Hum Genet . 2016; 24(12): 1730–1738. Pakarinen L, Tuppurainen K, Laippala P, Mäntyjärvi M, Puhakka H. The ophthalmological course of Usher syndrome type III. Int Ophthalmol . 1995; 19(5): 307–311. Khalaileh A, Abu-Diab A, Ben-Yosef T, et al. The genetics of Usher syndrome in the Israeli and Palestinian populations. Invest Ophthalmol Vis Sci . 2018; 59(2): 1095–1104. Fields RR, Zhou G, Huang D, et al. Usher syndrome type III: revised genomic structure of the USH3 gene and identification of novel mutations. Am J Hum Genet . 2002; 71(3): 607–617. Ness SL, Ben-Yosef T, Bar-Lev A, et al. Genetic homogeneity and phenotypic variability among Ashkenazi Jews with Usher syndrome type III. J Med Genet . 2003; 40(10): 767–772. Herrera W, Aleman TS, Cideciyan AV, et al. Retinal disease in Usher syndrome III caused by mutations in the clarin-1 gene. Invest Ophthalmol Vis Sci . 2008; 49(6): 2651. Ratnam K, Västinsalo H, Roorda A, Sankila E-MK, Duncan JL. Cone structure in patients with usher syndrome type III and mutations in the Clarin 1 gene. JAMA Ophthalmol . 2013; 131(1): 67–74. Audo I, Friedrich A, Mohand-Saïd S, Lancelot M-E, Antonio A, Moskova-Doumanova V, Poch O, Bhattacharya S, Sahel J-A, Zeitz C. An unusual retinal phenotype associated with a novel mutation in RHO. Arch Ophthalmol . 2010; 128(8): 1036–1045. Joensuu T, Hämäläinen R, Yuan B, et al. Mutations in a novel gene with transmembrane domains underlie Usher syndrome type 3. Am J Hum Genet . 2001; 69(4): 673–684. Ebermann I, Lopez I, Bitner-Glindzicz M, Brown C, Koenekoop RK, Bolz HJ. Deafblindness in French Canadians from Quebec: a predominant founder mutation in the USH1C gene provides the first genetic link with the Acadian population. Genome Biol . 2007; 8(4): R47. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med . 2015; 17(5): 405–424. Pakarinen L, Karjalainen S, Simola KOJ, Laippala P, Kaitalo H. Usher's syndrome type 3 in Finland. Laryngoscope . 1995; 105(6): 613–617. Plantinga RF, Pennings RJE, Huygen PLM, et al. Visual impairment in Finnish Usher syndrome type III. Acta Ophthalmol Scand . 2006; 84(1): 36–41. Duncker T, Tabacaru MR, Lee W, Tsang SH, Sparrow JR, Greenstein VC. Comparison of near-infrared and short-wavelength autofluorescence in retinitis pigmentosa. Invest Ophthalmol Vis Sci . 2013; 54(1): 585–591. Nassisi M, Lavia C, Mohand-Said S, et al. Near-infrared fundus autofluorescence alterations correlate with swept-source optical coherence tomography angiography findings in patients with retinitis pigmentosa. Sci Rep . 2021; 11(1): 3180. Ebermann I, Wilke R, Lauhoff T, Lübben D, Zrenner E, Bolz HJ. Two truncating USH3A mutations, including one novel, in a German family with Usher syndrome. Mol Vis . 2007; 13: 1539–1547. Västinsalo H, Jalkanen R, Dinculescu A, et al. Alternative splice variants of the USH3A gene Clarin 1 ( CLRN1 ). Eur J Hum Genet . 2011; 19(1): 30–35. Zallocchi M, Meehan DT, Delimont D, et al. Localization and expression of clarin-1 , the Clrn1 gene product, in auditory hair cells and photoreceptors. Hear Res . 2009; 255(1–2): 109–120. Xu L, Bolch SN, Santiago CP, et al. Clarin-1 expression in adult mouse and human retina highlights a role of Müller glia in Usher syndrome. J Pathol . 2020; 250(2): 195–204. Geller SF, Guerin KI, Visel M, et al. CLRN1 is nonessential in the mouse retina but is required for cochlear hair cell development. PLoS Genet ; 5(8): e1000607. Sahly I, Dufour E, Schietroma C, et al. Localization of Usher 1 proteins to the photoreceptor calyceal processes, which are absent from mice. J Cell Biol . 2012; 199(2): 381–399. Dinculescu A, Stupay RM, Deng W-T, et al. AAV-mediated clarin-1 expression in the mouse retina: implications for USH3A gene therapy. PLoS One . 2016; 11(2): e0148874. Geng R, Melki S, Chen DH-C, et al. The mechanosensory structure of the hair cell requires clarin-1, a protein encoded by Usher syndrome III causative gene. J Neurosci . 2012; 32(28): 9485–9498. Tian G, Zhou Y, Hajkova D, et al. Clarin-1, encoded by the Usher syndrome III causative gene, forms a membranous microdomain: possible role of clarin-1 in organizing the actin cytoskeleton. J Biol Chem . 2009; 284(28): 18980–18993. Geng R, Omar A, Gopal SR, et al. Modeling and preventing progressive hearing loss in Usher syndrome III. Sci Rep . 2017; 7(1): 13480. Geng R, Geller SF, Hayashi T, et al. Usher syndrome IIIA gene clarin-1 is essential for hair cell function and associated neural activation. Hum Mol Genet . 2009; 18(15): 2748–2760.

Kandaswamy, S., Zobel, L., John, B. et al. | Cell Death Discovery | Vol 8 , Article 387 | 2022 | doi.org/10.1038/s41420-022-01185-0 Abstract Retinitis pigmentosa is a group of progressive inherited retinal dystrophies that may present clinically as part of a syndromic entity or as an isolated (nonsyndromic) manifestation. In an Indian family suffering from retinitis pigmentosa, we identified a missense variation in CNGA1 affecting the cyclic nucleotide binding domain (CNBD) and characterized a mouse model developed with mutated CNBD. A gene panel analysis comprising 105 known RP genes was used to analyze a family with autosomal-recessive retinitis pigmentosa (arRP) and revealed that CNGA1 was affected. From sperm samples of ENU mutagenesis derived F1 mice, we re-derived a mutant with a Cnga1 mutation. Homozygous mutant mice, developing retinal degeneration, were examined for morphological and functional consequences of the mutation. In the family, we identified a rare CNGA1 variant (NM_001379270.1) c.1525 G > A; (p.Gly509Arg), which co-segregated among the affected family members. Homozygous Cnga1 mice harboring a (ENSMUST00000087213.12) c.1526 A > G (p.Tyr509Cys) mutation showed progressive degeneration in the retinal photoreceptors from 8 weeks on. This study supports a role for CNGA1 as a disease gene for arRP and provides new insights on the pathobiology of cGMP-binding domain mutations in CNGA1 -RP. Introduction Retinitis pigmentosa (RP) is a group of Inherited Retinal Degeneration/Dystrophies (IRD), with a global prevalence of 1 in 3000–7000 [ 1 ]. RP is characterized by abnormalities in the photoreceptors (rods and cones) or the retinal pigment epithelium (RPE) with all types of inheritance patterns documented. RP can occur either as isolated or as syndrome with the involvement of other organs such as the associated hearing loss in USHER syndrome. About 90 genes are known until date to cause RP (Retnet database http://www.sph.uth.tmc.edu/retnet/ ). Most of the gene variants in RP are directly associated with the phototransduction cascade, such as RHO (rhodopsin), which are known to cause 25–30% of adRP. Phototransduction begins with the detection of light photons by rhodopsin, and this triggers several signaling steps that eventually convert the light signal into an electrical signal being transmitted to the brain. Key steps of this downstream signaling are mediated by proteins encoded by genes linked to RP. This list includes genes encoding for the subunits of rod phosphodiesterase ( PDE6A and PDE6B ) and rod cyclic nucleotide gated (CNG) channel ( CNGA1 and CNGB1 ). CNGA1 encodes the A (or alpha) subunit of the rod CNG channel, which is a heterotetrameric channel complex formed by three CNGA1 and one CNGB1 subunits; its structure has been recently solved [ 2 ]. The rod CNG channel along with CNGB1 forms a cyclic guanosine monophosphate (cGMP)-gated cation channel found in the rod photoreceptor outer segment plasma membrane [ 3 ]. Each CNG channel subunit consist of six transmembrane domains, and both, the N-terminal and the C-terminal domain, are in the cytoplasm [ 2 ]. While the A subunit is essential for the principle formation of a functional cGMP-gated channel, the B subunit is important for transport of the channel to the plasma membrane of the rod outer segment and confers specific properties to the channel complex such as rapid on-off kinetics and sensitivity to the pharmacological inhibitor L-cis-diltiazem [ 4 , 5 ]. In the present study we report a rare variant (c.1525 G > A; p.Gly509Arg) of the CNGA1 gene in a family suffering from arRP. Animal models for retinal degeneration usually provide insights into pathological mechanism of disease progression and assist in designing therapeutic strategies. We re-derived a Cnga1 (c1526A > G; pTyr509Cys) mouse mutant from the ENU archive [ 6 ]. This mutation falls within the same protein domain as the one observed in the human family. We report herein the retinal degeneration by a longitudinal morphological and physiological analysis.

Juan L. López-Cánovas , Natalia Hermán-Sánchez , Mercedes del Rio-Moreno , Antonio C. Fuentes-Fayos , Araceli Lara-López , Marina E. Sánchez-Frias , Víctor Amado , Rubén Ciria , Javier Briceño , Manuel de la Mata , Justo P. Castaño , Manuel Rodriguez-Perálvarez , Raúl M. Luque , Manuel D. Gahete  | Experimental & Molecular Medicine | 06 January 2023 | Volume 55 | Pages132–142 Abstract Hepatocellular carcinoma (HCC) pathogenesis is associated with alterations in splicing machinery components (spliceosome and splicing factors) and aberrant expression of oncogenic splice variants. We aimed to analyze the expression and potential role of the spliceosome component PRPF8 (pre-mRNA processing factor 8) in HCC. PRPF8 expression (mRNA/protein) was analyzed in a retrospective cohort of HCC patients ( n  = 172 HCC and nontumor tissues) and validated in two in silico cohorts (TCGA and CPTAC). PRPF8 expression was silenced in liver cancer cell lines and in xenograft tumors to understand the functional and mechanistic consequences. In silico RNAseq and CLIPseq data were also analyzed. Our results indicate that PRPF8 is overexpressed in HCC and associated with increased tumor aggressiveness (patient survival, etc.), expression of HCC-related splice variants, and modulation of critical genes implicated in cancer-related pathways. PRPF8 silencing ameliorated aggressiveness in vitro and decreased tumor growth in vivo. Analysis of in silico CLIPseq data in HepG2 cells demonstrated that PRPF8 binds preferentially to exons of protein-coding genes, and RNAseq analysis showed that PRPF8 silencing alters splicing events in multiple genes. Integrated and in vitro analyses revealed that PRPF8 silencing modulates fibronectin (FN1) splicing, promoting the exclusion of exon 40.2, which is paramount for binding to integrins. Consistent with this finding, PRPF8 silencing reduced FAK/AKT phosphorylation and blunted stress fiber formation. Indeed, HepG2 and Hep3B cells exhibited a lower invasive capacity in membranes treated with conditioned medium from PRPF8 -silenced cells compared to medium from scramble-treated cells. This study demonstrates that PRPF8 is overexpressed and associated with aggressiveness in HCC and plays important roles in hepatocarcinogenesis by altering FN1 splicing, FAK/AKT activation and stress fiber formation. Introduction Hepatocellular carcinoma (HCC) is the most prevalent type of primary liver cancer and the fourth most common cancer worldwide 1 . The majority of HCC cases are associated with chronic liver diseases due to alcohol consumption, chronic viral hepatitis or metabolic syndrome, among other etiologies 1 . Since the 1990s, the incidence of HCC has increased dramatically, and HCC-related mortality is increasing faster than that of other cancer types 1 . The elevated death rates observed for HCC could be associated with its late diagnosis despite routine screening strategies with liver ultrasound every 6 months and because the available therapies have a limited impact on overall survival 2 . Therefore, a better understanding of hepatocellular carcinogenesis could help to identify new diagnostic, prognostic and therapeutic targets. A common hallmark of cancer is the alteration of important elements regulating cell physiology, especially the presence or aberrant expression of splice variants, which could be associated with development, progression and drug resistance in different types of cancer 3 . In fact, splice variants of important genes such as CDCC50 , KLF6 , and FN1 4 , 5 are involved in liver carcinogenesis, thus suggesting that an altered splicing process could play an essential role in the development and progression of HCC 6 . The splicing process is controlled by the spliceosome, a macromolecular ribonucleoprotein complex that cooperates with hundreds of splicing factors to catalyze this process 7 . This splicing machinery is essential for appropriate modulation of gene expression, and its dysregulation is associated with oncogenic progression, including in HCC 8 , and with the generation of an aberrant landscape of alternative splice variants 9 . In this context, the splicing factor PRPF8 (pre-mRNA processing factor 8) is a key protein in the catalytic nucleus of the spliceosome, which participates in the second step of the splicing process. In Drosophila , loss of PRPF8 decreases cell proliferation, increases cell death and modulates cell differentiation and polarity, and PRPF8-mediated hyperplastic growth is induced by different oncogenes 10 . Indeed, PRPF8 seems to be crucial for appropriate constitutive and alternative mRNA splicing 11 . PRPF8 mutations have been associated with severe forms of retinitis pigmentosa as well as with the initiation of various types of myeloid neoplasms and with decreased survival in patients with leukemia 12 . In solid tumors, silencing of PRPF8 was found to result in cancer subtype-specific implications in breast cancer cell lines 13 , while in prostate cancer, PRPF8 is involved in androgen receptor splicing 14 . In addition, recent evidence suggests that PRPF8 may also be overexpressed in HCC and be associated with the tumorigenic potential; however, these conclusions were based on a single HCC cohort and experiments with a single HCC cell line 15 . Based on this information, and using several in vitro approaches, animal models, and human samples, we aimed to explore the putative dysregulation, association with clinical parameters and functional role of PRPF8 in an ample number of HCC cohorts and cell lines; the implication of PRPF8 in the control of the splicing process in HCC; and the potential utility of its genetic modulation in hepatocarcinogenesis. Click here to read full article References Villanueva, A. Hepatocellular Carcinoma. N. Engl. J. Med. 380 , 1450–1462 (2019). Forner, A., Reig, M. & Bruix, J. Hepatocellular carcinoma. Lancet 391 , 1301–1314 (2018). Sveen, A., Kilpinen, S., Ruusulehto, A., Lothe, R. A. & Skotheim, R. I. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes. Oncogene 35 , 2413–2427 (2016). Wang, H. et al. A Coiled-Coil Domain Containing 50 Splice Variant Is Modulated by Serine/Arginine-Rich Splicing Factor 3 and Promotes Hepatocellular Carcinoma in Mice by the Ras Signaling Pathway. Hepatology 69 , 179–195 (2019).   Vetter, D. et al. Enhanced hepatocarcinogenesis in mouse models and human hepatocellular carcinoma by coordinate KLF6 depletion and increased messenger RNA splicing. Hepatology 56 , 1361–1370 (2012).   Lee, S. E., Alcedo, K. P., Kim, H. J. & Snider, N. T. Alternative Splicing in Hepatocellular Carcinoma. Cell Mol. Gastroenterol. Hepatol. 10 , 699–712 (2020). Matera, A. G. & Wang, Z. A day in the life of the spliceosome. Nat. Rev. Mol. Cell. Biol. 15 , 108–121 (2014). Lopez-Canovas, J. L. et al. Splicing factor SF3B1 is overexpressed and implicated in the aggressiveness and survival of hepatocellular carcinoma. Cancer Lett. 496 , 72–83 (2021). Kelemen, O. et al. Function of alternative splicing. Gene 514 , 1–30 (2013). Fernandez-Espartero, C. H. et al. Prp8 regulates oncogene-induced hyperplastic growth in Drosophila. Development 145 , dev162156 (2018). Wickramasinghe, V. O. et al. Regulation of constitutive and alternative mRNA splicing across the human transcriptome by PRPF8 is determined by 5’ splice site strength. Genome Biol. 16 , 201 (2015). Kurtovic-Kozaric, A. et al. PRPF8 defects cause missplicing in myeloid malignancies. Leukemia 29 , 126–136 (2015). Neelamraju, Y., Gonzalez-Perez, A., Bhat-Nakshatri, P., Nakshatri, H. & Janga, S. C. Mutational landscape of RNA-binding proteins in human cancers. RNA Biol. 15 , 115–129 (2018). Wang, D. et al. Splicing Factor Prp8 Interacts With NES(AR) and regulates androgen receptor in prostate cancer cells. Mol. Endocrinol. 29 , 1731–1742 (2015).

H. Lorach , S. Kang , R. Dalal , M. B. Bhuckory , Y. Quan , D. Palanker  | Scientific Reports | 27 July 2018 | https://doi.org/10.1038/s41598-018-29631-z Abstract MERTK mutation reduces the ability of retinal pigment epithelial (RPE) cells to phagocytize the photoreceptor outer segments, which leads to accumulation of debris separating photoreceptors from RPE cells, resulting in their degeneration and loss of vision. In a rat model of Retinitis Pigmentosa due to MERTK mutation, we demonstrate that surgical removal of debris performed when about half of photoreceptors are lost (P38), allows the remaining photoreceptor cells to renew their outer segments and survive for at least 6 months – 3 times longer than in untreated eyes. In another set of experiments, patterned laser photocoagulation was performed before the debris formation (P19-25) to destroy a fraction of photoreceptors and thereby reduce the phagocytic load of shed outer segment fragments. This treatment also delayed the degeneration of the remaining photoreceptors. Both approaches were assessed functionally and morphologically, using electroretinography, optical coherence tomography, and histology. The long-term preservation of photoreceptors we observed indicates that MERTK -related form of inherited retinal degeneration, which has currently no cure, could be amenable to laser therapy or subretinal surgery, to extend the visual function, potentially for life. Introduction Retinitis pigmentosa (RP) is a group of inherited retinal diseases that can lead to profound loss of vision and eventual blindness due to progressive degeneration of photoreceptor cells. These disorders can be caused by a wide variety of genetic defects. To date, nearly 4300 mutations in 79 genes have been reported to cause RP 1 . Many of the associated genes encode proteins that are involved in phototransduction, photoreceptor structure, or photoreceptor gene transcription. MER-proto-oncogene, tyrosine kinase ( MERTK ) gene encodes for a transmembrane protein involved in recognition and phagocytosis of the photoreceptor outer segments, essential for recycling of the phototransduction machinery. Mutations in the MERTK gene cause reduced phagocytic function, which leads to accumulation of photoreceptor outer segment debris in subretinal space. This debris subsequently impedes efficient oxygen and nutrient transport to photoreceptor cells. This mutation was actually identified thanks to a spontaneous rodent model of retinal degeneration: the Royal College of Surgeons (RCS) rat. This first animal model of inherited retinal degeneration, was described by Bourne and coworkers in 1938 2 . Since 1970s, it has been known that the RCS rat has dysfunctional RPE cells, which are unable to phagocytize shed outer segment fragments, leading to their accumulation as a debris between photoreceptors and RPE cells 3 , 4 . Recently, mutations in the MERTK gene were identified as the cause of the functional defect in RPE cells of RCS rat 5 . In humans, mutations of the MERTK gene lead to a loss of night vision in early childhood, gradual constriction of the visual field, and eventual loss of visual acuity before adulthood. Imaging studies using optical coherence tomography (OCT) in these patients revealed the loss of photoreceptors and formation of a hyper-reflective layer, analogous to the debris layer in RCS rats 6 , 7 . In the RCS rats, appearance of the outer segment debris begins around postnatal day (P) 19. These debris accumulate with age 8 , forming a thick insulating layer between photoreceptors and RPE cells by P35, accompanied by gradual degeneration of photoreceptors, which is complete by P180. RCS animal model is widely used today in research of retinal degeneration, including strategies for RPE cell transplantation 9 , 10 , 11 , 12 , 13 , 14 , gene therapy 15 , 16 , 17 , 18 or retinal prosthetics 19 , 20 , 21 , 22 , 23 . Many of these studies do not introduce a proper sham procedure group, while a few that do, often report a protective effect in the sham surgery group, but tend to undermine and qualify it as a short-term effect 24 . One study 25 , focused on the photoreceptors rescue effect by retinal detachment, demonstrated anatomical rescue comparable to results with RPE transplantation. Unfortunately, this study followed the animals only for 2 months and was not designed to show functional preservation of vision. Since retinal outer segments are shed daily, but debris accumulation does not start for a few years in patients and for a few weeks in RCS rats, while retina continues to function long after that, we hypothesized that the mutant RPE cells retain some phagocytic activity either by MERTK -independent uptake or microglia-related phagocytosis 26 , and thereby can sustain a fraction of the outer-segment recycling load 27 . We also assumed that photoreceptors degeneration is accelerated by accumulation of a thick debris layer, which prevents oxygen and nutrients supply from the choroid. Based on these hypotheses, we designed two strategies to balance the supply and demand of the outer segment recycling and thereby extend the survival of photoreceptors in RCS rats. Both strategies utilize clinically readily available techniques. First, debris layer can be surgically washed out from subretinal space when a substantial fraction of functional photoreceptors is still present. Alternatively, pattern laser photocoagulation can be applied to selectively destroy a fraction of photoreceptors prior to debris formation. In both approaches, the remaining photoreceptors could be better sustained by the partially functioning RPE cells and survive much longer. In this study, we assess efficacy of these therapies for protection of photoreceptors in RCS rats as a potential therapy for patients with RP due to MERTK mutation. Click here to read entire paper References Daiger, S. P., Sullivan, L. S. & Bowne, S. J. Chapter 31 - Genetic Mechanisms of Retinal Disease. in Retina (Fifth Edition) (ed. Schachat, S. J. R. R. S. R. H. P. S. R. S. P. W. W. P.) 624–634 (W. B. Saunders, London, 2013). Bourne, M. C., Campbell, D. A. & Tansley, K. Hereditary Degeneration of the Rat Retina. Br J Ophthalmol 22, 613–623 (1938). Bok, D. & Hall, M. O. The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat. J Cell Biol 49, 664–682 (1971).  Mullen, R. J. & LaVail, M. M. Inherited retinal dystrophy: primary defect in pigment epithelium determined with experimental rat chimeras. Science 192, 799–801 (1976). D’Cruz, P. M. et al . Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat. Human molecular genetics 9, 645–651 (2000). Charbel Issa, P. et al . Characterisation of severe rod-cone dystrophy in a consanguineous family with a splice site mutation in the MERTK gene. Br J Ophthalmol 93, 920–925 (2009). Mackay, D. S. et al . Novel mutations in MERTK associated with childhood onset rod-cone dystrophy. Molecular vision 16, 369–377 (2010). Dowling, J. E. & Sidman, R. L. Inherited retinal dystrophy in the rat. J Cell Biol 14, 73–109 (1962). Sauve, Y., Klassen, H., Whiteley, S. J. & Lund, R. D. Visual field loss in RCS rats and the effect of RPE cell transplantation. Experimental neurology 152, 243–250 (1998). Sheedlo, H. J., Li, L. X. & Turner, J. E. Functional and structural characteristics of photoreceptor cells rescued in RPE-cell grafted retinas of RCS dystrophic rats. Experimental eye research 48, 841–854 (1989). Wang, S. et al . Morphological and functional rescue in RCS rats after RPE cell line transplantation at a later stage of degeneration. Investigative ophthalmology & visual science 49, 416–421 (2008). Thomas, B. B. et al . Survival and Functionality of hESC-Derived Retinal Pigment Epithelium Cells Cultured as a Monolayer on Polymer Substrates Transplanted in RCS Rats. Investigative ophthalmology & visual science 57, 2877–2887 (2016). Coffey, P. J. et al . Long-term preservation of cortically dependent visual function in RCS rats by transplantation. Nature neuroscience 5, 53–56 (2002). Kole, C. et al . Otx2-genetically modified retinal pigment epithelial cells rescue photoreceptors after transplantation. Molecular Therapy (2017). Vollrath, D. et al . Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk. Proceedings of the National Academy of Sciences of the United States of America 98, 12584–12589 (2001). Tschernutter, M. et al . Long-term preservation of retinal function in the RCS rat model of retinitis pigmentosa following lentivirus-mediated gene therapy. Gene therapy 12, 694–701 (2005). Deng, W. T. et al . Tyrosine-mutant AAV8 delivery of human MERTK provides long-term retinal preservation in RCS rats. Investigative ophthalmology & visual science 53, 1895–1904 (2012). LaVail, M. M. et al . Gene Therapy for MERTK-Associated Retinal Degenerations. Adv Exp Med Biol 854, 487–493 (2016). Ciavatta, V. T., Mocko, J. A., Kim, M. K. & Pardue, M. T. Subretinal electrical stimulation preserves inner retinal function in RCS rat retina. Molecular vision 19, 995–1005 (2013). Alamusi, Matsuo, T., Hosoya, O. & Uchida, T. Visual evoked potential in RCS rats with Okayama University-type retinal prosthesis (OUReP) implantation. Journal of artificial organs: the official journal of the Japanese Society for Artificial Organs 20, 158–165 (2017). Maya-Vetencourt, J. F. et al . A fully organic retinal prosthesis restores vision in a rat model of degenerative blindness. Nature materials 16, 681–689 (2017). Lorach, H. et al . Photovoltaic restoration of sight with high visual acuity. Nature medicine (2015). Smith, A. J. et al . AAV-Mediated gene transfer slows photoreceptor loss in the RCS rat model of retinitis pigmentosa. Molecular therapy: the journal of the American Society of Gene Therapy 8, 188–195 (2003). Lu, B. et al . Neural Stem Cells Derived by Small Molecules Preserve Vision. Translational vision science & technology 2, 1 (2013). Silverman, M. S. & Hughes, S. E. Photoreceptor rescue in the RCS rat without pigment epithelium transplantation. Current eye research 9, 183–191 (1990). Palczewska, G. et al . Receptor MER Tyrosine Kinase Proto-oncogene (MERTK) Is Not Required for Transfer of Bis-retinoids to the Retinal Pigmented Epithelium. J Biol Chem 291, 26937–26949 (2016). Chaitin, M. H. & Hall, M. O. Defective ingestion of rod outer segments by cultured dystrophic rat pigment epithelial cells. Investigative ophthalmology & visual science 24, 812–820 (1983). Sher, A. et al . Restoration of retinal structure and function after selective photocoagulation. J Neurosci 33, 6800–6808 (2013). Paulus, Y. M. et al . Healing of retinal photocoagulation lesions. Investigative ophthalmology & visual science 49, 5540–5545 (2008).  McGill, T. J. et al . Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation. Translational vision science & technology 6, 17 (2017). Qu, L. et al . Combined transplantation of human mesenchymal stem cells and human retinal progenitor cells into the subretinal space of RCS rats. Scientific reports 7, 199 (2017). Ghazi, N. G. et al . Treatment of retinitis pigmentosa due to MERTK mutations by ocular subretinal injection of adeno-associated virus gene vector: results of a phase I trial. Human genetics 135, 327–343 (2016). Photocoagulation treatment of proliferative diabetic retinopathy. Clinical application of Diabetic Retinopathy Study (DRS) findings, DRS Report Number 8. The Diabetic Retinopathy Study Research Group. Ophthalmology 88, 583–600 (1981). Treatment techniques and clinical guidelines for photocoagulation of diabetic macular edema. Early Treatment Diabetic Retinopathy Study Report Number 2. Early Treatment Diabetic Retinopathy Study Research Group. Ophthalmology 94, 761–774 (1987). Hughes, A. A schematic eye for the rat. Vision research 19, 569–588 (1979). McLaren, M. J. & Inana, G. Inherited retinal degeneration: basic FGF induces phagocytic competence in cultured RPE cells from RCS rats. FEBS letters 412, 21–29 (1997). Wen, R. et al . Injury-induced upregulation of bFGF and CNTF mRNAS in the rat retina. J Neurosci 15, 7377–7385 (1995). Cao, W., Wen, R., Li, F., Lavail, M. M. & Steinberg, R. H. Mechanical injury increases bFGF and CNTF mRNA expression in the mouse retina. Experimental eye research 65, 241–248 (1997). Cao, W., Li, F., Steinberg, R. H. & Lavail, M. M. Development of normal and injury-induced gene expression of aFGF, bFGF, CNTF, BDNF, GFAP and IGF-I in the rat retina. Experimental eye research 72, 591–604 (2001). Humphrey, M. F., Chu, Y., Mann, K. & Rakoczy, P. Retinal GFAP and bFGF expression after multiple argon laser photocoagulation injuries assessed by both immunoreactivity and mRNA levels. Experimental eye research 64, 361–369 (1997). Carwile, M. E., Culbert, R. B., Sturdivant, R. L. & Kraft, T. W. Rod outer segment maintenance is enhanced in the presence of bFGF, CNTF and GDNF. Experimental eye research 66, 791–805 (1998). Ozaki, S., Radeke, M. J. & Anderson, D. H. Rapid upregulation of fibroblast growth factor receptor 1 (flg) by rat photoreceptor cells after injury. Investigative ophthalmology & visual science 41, 568–579 (2000). Chu, Y., Humphrey, M. F., Alder, V. V. & Constable, I. J. Immunocytochemical localization of basic fibroblast growth factor and glial fibrillary acidic protein after laser photocoagulation in the Royal College of Surgeons rat. Australian and New Zealand journal of ophthalmology 26, 87–96 (1998). Ksantini, M., Lafont, E., Bocquet, B., Meunier, I. & Hamel, C. P. Homozygous mutation in MERTK causes severe autosomal recessive retinitis pigmentosa. European journal of ophthalmology 22, 647–653 (2012). Lorach, H. et al . Development of Animal Models of Local Retinal Degeneration. Investigative ophthalmology & visual science 56, 4644–4652 (2015).

Federica E. Poli, Imran H. Yusuf, Penny Clouston, Morag Shanks, Jennifer Whitfield, Peter Charbel Issa, Robert E. MacLaren | Ophthalmic Genetics | 29 Aug 2022 | 44 (1) | pgs. 74-82 | doi.org/10.1080/13816810.2022.2113541 Abstract Background MERTK (MER proto-oncogene, tyrosine kinase) is a transmembrane protein essential in regulating photoreceptor outer segment phagocytosis. Biallelic mutations in MERTK cause retinal degeneration. Here we present the retinal phenotype of three patients with missense variants in MERTK . Materials and methods All patients underwent a full clinical examination, fundus photography, short-wavelength fundus autofluorescence and optical coherence tomography imaging. Two patients also underwent Goldmann visual field testing and electroretinography was undertaken for the third patient. Molecular genetic testing was undertaken using next generation or whole-exome sequencing with all variants confirmed by Sanger sequencing. Results The first patient was a 29-year-old female heterozygous for a missense variant (c.1133C>T, p.Thr378 Met) and a nonsense variant (c.1744_1751delinsT, p.Ile582Ter) in MERTK . The second patient was a 26-year-old male homozygous for a c.2163T>A, p.His721Gln variant in MERTK . The third patient was an 11-year-old female heterozygous for a deletion of exons 5–19 and a missense variant (c.1866 G>C, p.Lys622Asn) in MERTK . Reduced night vision was the initial symptom in all patients. Fundoscopy revealed typical signs of retinitis pigmentosa (RP) with early-onset macular atrophy. All three MERTK missense variants affect highly conserved residues within functional domains, have low population frequencies and are predicted to be pathogenic in silico . Conclusions We report three missense variants in MERTK and present the associated phenotypic data, which are supportive of non-syndromic RP. MERTK is a promising candidate for viral-mediated gene replacement therapy. Moreover, one variant represents a single nucleotide transition, which is theoretically targetable with CRISPR-Cas9 base-editing. Introduction Retinitis pigmentosa (RP) is a set of clinically and genetically heterogenous inherited retinal dystrophies characterised by progressive primary rod photoreceptor degeneration with concurrent, or later degeneration of cones ( Citation1 ). It typically manifests with difficulties in dark adaptation and night vision, followed by progressive peripheral visual field loss, with subsequent loss of central vision. A vast number of heterogenous genetic defects have been implicated in the pathogenesis of non-syndromic RP ( Citation2 , Citation3 ). The MER proto-oncogene, tyrosine kinase ( MERTK ) gene encodes a transmembrane protein expressed in the retinal pigment epithelium (RPE), which plays a critical role in photoreceptor homeostasis by regulation of phagocytosis of shed photoreceptor outer segment discs ( Figure 1 , panel A) ( Citation4 , Citation5 ). Numerous mutations in MERTK have been identified as pathogenic for retinal dystrophies ( Citation6 , Citation7 ). MERTK mutations cause a rod-cone dystrophy with early macular atrophy, with RP being the most common retinal phenotype, although cases of Leber congenital amaurosis have been reported ( Citation7 ). Figure 1. Panel A: structure of the MERTK transmembrane protein. The extracellular portion includes two immunoglobulin-like (Ig-like) domains (green) and two fibronectin type III (FN-III) domains (blue). The intracellular region contains a highly conserved kinase domain (yellow). The location of the Three mutations discussed are indicated by red arrows. The respective amino acid residues corresponding to the domains affected are indicated below the protein schematic. Panel B: Conservation across species of the amino acid residue subject to mutation in the c.1133c>t, p.Thr378 met variant (B1), the c.2163t>a, p.His721gln variant (B2) and the c.1866G>C, p.Lys622Asn variant (B3). Panel C: Pedigrees for Case 1 (C1) and Case 2 (C2). Pedigree not available for Case 3. Note the pedigrees are based on clinical presentation and segregation has not been possible. Click here to read entire paper References Hartong DT, Berson EL, Dryja TP. Retinitis pigmentosa. Lancet. 2006;368(9549):1795–809. doi:10.1016/S0140-6736(06)69740-7. RetNet: summaries of genes and loci causing retinal diseases. 2021. https://sph.uth.edu/retnet/sum-dis.htm#A-genes(open in a new window) . Verbakel SK, van Huet RA, Boon CJ, den Hollander AI, Collin RW, Klaver CC, Hoyng CB, Roepman R, Klevering BJ. Non-syndromic retinitis pigmentosa. Prog Retin Eye Res. 2018;66:157–86. Feng W, Yasumura D, Matthes MT, LaVail MM, Vollrath D. Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells. J Biol Chem. 2002;277(19):17016–22. doi:10.1074/jbc.M107876200. Lemke G. Biology of the TAM receptors. Cold Spring Harb Perspect Biol. 2013;5(11):a009076. doi:10.1101/cshperspect.a009076. Gal A, Li Y, Thompson DA, Weir J, Orth U, Jacobson SG, Apfelstedt-Sylla E, Vollrath D. Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa. Nat Genet. 2000;26(3):270–71. doi:10.1038/81555. Audo I, Mohand‐said S, Boulanger‐scemama E, Zanlonghi X, Condroyer C, Démontant V, Boyard F, Antonio A, Méjécase C, El Shamieh S, Sahel JA. MERTK mutation update in inherited retinal diseases. Hum Mutat. 2018;39(7):887–913. doi:10.1002/humu.23431. Ellingford JM, Barton S, Bhaskar S, O’Sullivan J, Williams SG, Lamb JA, Panda B, Sergouniotis PI, Gillespie RL, Daiger SP, et al. Molecular findings from 537 individuals with inherited retinal disease. J Med Genet. 2016;53(11):761–67. doi:10.1136/jmedgenet-2016-103837. Takahashi VK, Xu CL, Takiuti JT, Apatoff MBL, Duong JK, Mahajan VB, Tsang SH. Comparison of structural progression between ciliopathy and non-ciliopathy associated with autosomal recessive retinitis pigmentosa. Orphanet J Rare Dis. 2019;14(1):1–9. doi:10.1186/s13023-019-1163-9. Colombo L, Maltese PE, Castori M, El Shamieh S, Zeitz C, Audo I, Zulian A, Marinelli C, Benedetti S, Costantini A, et al. Molecular epidemiology in 591 Italian probands with nonsyndromic retinitis pigmentosa and usher syndrome. Invest Ophthalmol Vis Sci. 2021;62(2):13. doi:10.1167/iovs.62.2.13. Shanks ME, Downes SM, Copley RR, Lise S, Broxholme J, Hudspith KA, Kwasniewska A, Davies WI, Hankins MW, Packham ER, et al. Next-generation sequencing (NGS) as a diagnostic tool for retinal degeneration reveals a much higher detection rate in early-onset disease. Eur J Hum Genet. 2013;21(3):274–80. doi:10.1038/ejhg.2012.172. D’Cruz PM, Yasumura D, Weir J, Matthes MT, Abderrahim H, LaVail MM, Vollrath D. Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat. Hum Mol Genet. 2000;9(4):645–51. doi:10.1093/hmg/9.4.645. Nandrot E, Dufour EM, Provost AC, Péquignot MO, Bonnel S, Gogat K, Marchant D, Rouillac C, Sépulchre de Condé B, Bihoreau M-T, et al. Homozygous deletion in the coding sequence of the c-mer gene in RCS rats unravels general mechanisms of physiological cell adhesion and apoptosis. Neurobiol Dis. 2000;7(6):586–99. doi:10.1006/nbdi.2000.0328. Mullen RJ, LaVail MM. Inherited retinal dystrophy: primary defect in pigment epithelium determined with experimental rat chimeras. Science. 1976;192(4241):799–801. doi:10.1126/science.1265483. Dowling JE, Sidman RL. Inherited retinal dystrophy in the rat. J Cell Biol. 1962;14(1):73–109. doi:10.1083/jcb.14.1.73. Bok D, Hall MO. The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat. J Cell Biol. 1971;49(3):664–82. doi:10.1083/jcb.49.3.664. Jonsson F, Burstedt M, Kellgren TG, Golovleva I Non-Homologous recombination between Alu and LINE-1 repeats results in a 91 kb deletion in MERTK causing severe retinitis pigmentosa. Mol Vis. 2018;24:667. Liu S, Bi JG, Hu Y, Tang D, Li B, Zhu P, Peng W, Du D, He H, Zeng J, et al. Targeted next generation sequencing identified novel loss-of-function mutations in MERTK gene in Chinese patients with retinitis pigmentosa. Mol Genet Genomic Med. 2019;7(4):e00577. doi:10.1002/mgg3.577. Jespersgaard C, Bertelsen M, Arif F, Gellert-Kristensen HG, Fang M, Jensen H, Rosenberg T, Tümer Z, Møller LB, Brøndum-Nielsen K, et al. Bi-allelic pathogenic variations in MERTK including deletions are associated with an early onset progressive form of retinitis pigmentosa. Genes. 2020;11(12):1517. doi:10.3390/genes11121517. Birtel J, Eisenberger T, Gliem M, Müller PL, Herrmann P, Betz C, Zahnleiter D, Neuhaus C, Lenzner S, Holz FG, et al. Clinical and genetic characteristics of 251 consecutive patients with macular and cone/cone-rod dystrophy. Sci Rep. 2018;8(1):1–11. doi:10.1038/s41598-018-22096-0. Issa PC, Bolz HJ, Ebermann I, Domeier E, Holz FG, Scholl HP. Characterisation of severe rod–cone dystrophy in a consanguineous family with a splice site mutation in the MERTK gene. Br J Ophthalmol. 2009;93(7):920–25. doi:10.1136/bjo.2008.147397. Gliem M, Müller PL, Birtel J, Herrmann P, McGuinness MB, Holz FG, Charbel Issa P. Quantitative fundus autofluorescence and genetic associations in macular, cone, and cone–rod dystrophies. Ophthalmol Retina. 2020;4(7):737–49. doi:10.1016/j.oret.2020.02.009. Strick DJ, Vollrath D. Focus on molecules: MERTK. Exp Eye Res. 2010;91(6):786. doi:10.1016/j.exer.2010.05.006. Eisenberger T, Neuhaus C, Khan AO, Decker C, Preising MN, Friedburg C, Bieg A, Gliem M, Issa PC, Holz FG, et al. Increasing the yield in targeted next-generation sequencing by implicating CNV analysis, non-coding exons and the overall variant load: the example of retinal dystrophies. PloS One. 2013;8(11):e78496. doi:10.1371/journal.pone.0078496. Cehajic-Kapetanovic J, Xue K, de la Camara, A Nanda, Martinez-Fernandez C, Nanda LJ, Davies A, Wood, LJ, Fischer, MD, Aylward, JW, et al. Initial results from a first-in-human gene therapy trial on X-linked retinitis pigmentosa caused by mutations in RPGR. Nat Med. 2020;26(3):354–59. doi:10.1038/s41591-020-0763-1. Xue K, Jolly JK, Barnard AR, Rudenko A, Salvetti AP, Patrício MI, Edwards, TL, Groppe, M, Orlans, HO, Tolmachova, T, et al. Beneficial effects on vision in patients undergoing retinal gene therapy for choroideremia. Nat Med. 2018;24(10):1507–12. doi:10.1038/s41591-018-0185-5. Russell S, Bennett J, Wellman JA, Chung DC, Yu Z, Tillman A, Wittes, J, Pappas, J, Elci, O, McCague, S, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRpe65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet. 2017;390(10097):849–60. doi:10.1016/S0140-6736(17)31868-8. Askou AL, Jakobsen TS, Corydon TJ Retinal gene therapy: an eye-opener of the 21st century. Gene Ther. 2021;28(5):209–16. doi:10.1038/s41434-020-0168-2. Tschernutter M, Schlichtenbrede FC, Howe S, Balaggan KS, Munro PM, Bainbridge J, Thrasher, AJ, Smith, AJ, Ali, RR Long-Term preservation of retinal function in the RCS rat model of retinitis pigmentosa following lentivirus-mediated gene therapy. Gene Ther. 2005;12(8):694–701. doi:10.1038/sj.gt.3302460. Smith AJ, Schlichtenbrede FC, Tschernutter M, Bainbridge JW, Thrasher AJ, Ali RR AAV-Mediated gene transfer slows photoreceptor loss in the RCS rat model of retinitis pigmentosa. Mol Ther. 2003;8(2):188–95. doi:10.1016/S1525-0016(03)00144-8. Vollrath D, Feng W, Duncan JL, Yasumura D, D’Cruz PM, Chappelow A, Matthes, MT, Kay, MA, LaVail, MM Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk. Proc Nat Acad Sci. 2001;98(22):12584–89. doi:10.1073/pnas.221364198. Ghazi NG, Abboud EB, Nowilaty SR, Alkuraya H, Alhommadi A, Cai H, Hou, R, Deng, W-T, Boye, SL, Almaghamsi, A, et al. Treatment of retinitis pigmentosa due to MERTK mutations by ocular subretinal injection of adeno-associated virus gene vector: results of a phase I trial. Hum Genet. 2016;135(3):327–43. doi:10.1007/s00439-016-1637-y. Li T, Adamian M, Roof DJ, Berson EL, Dryja TP, Roessler BJ, Davidson, BL In vivo transfer of a reporter gene to the retina mediated by an adenoviral vector. Invest Ophthalmol Vis Sci. 1994;35(5):2543–49. Bennett J, Wilson J, Sun D, Forbes B, Maguire A Adenovirus vector-mediated in vivo gene transfer into adult murine retina. Invest Ophthalmol Vis Sci. 1994;35(5):2535–42. Dong J, Fan P, Frizzell RA Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther. 1996;7(17):2101–12. doi:10.1089/hum.1996.7.17-2101. Rees HA, Liu DR Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet. 2018;19(12):770–88. doi:10.1038/s41576-018-0059-1. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016;533(7603):420–24. doi:10.1038/nature17946. Cox DB, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang, F. RNA editing with CRISPR-Cas13. Science. 2017;358(6366):1019–27. doi:10.1126/science.aaq0180. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu, DR Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature. 2017;551(7681):464–71. doi:10.1038/nature24644. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen, PJ, Wilson, C, Newby, G A, Raguram, A., et al. Search-And-Replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576(7785):149–57. doi:10.1038/s41586-019-1711-4.

He, Y., Zhang, Y., Liu, X., Ghazaryan, E., Li, Y., Xie, J., & Su, G. (2014). Recent advances of stem cell therapy for retinitis pigmentosa. International journal of molecular sciences , 15 (8), 14456–14474. https://doi.org/10.3390/ijms150814456 Abstract Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by progressive loss of photoreceptors and eventually leads to retina degeneration and atrophy. Until now, the exact pathogenesis and etiology of this disease has not been clear, and many approaches for RP therapies have been carried out in animals and in clinical trials. In recent years, stem cell transplantation-based attempts made some progress, especially the transplantation of bone marrow-derived mesenchymal stem cells (BMSCs). This review will provide an overview of stem cell-based treatment of RP and its main problems, to provide evidence for the safety and feasibility for further clinical treatment. Introduction Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by progressive loss of photoreceptors and eventually leads to retina degeneration and atrophy. New approaches for RP therapies include: cell transplantation therapy, gene therapy, cytokine therapy, nutrition therapy, and hyperbaric oxygen therapy. Present therapies for RP are restricted in their efficacy or safety, for example: maintenance of long-term efficacy using a single injection of cytokines is difficult, but there is a risk of infection after repeated intra-vitreous injections in cytokine therapy. Gene therapy has been shown to improve visual function in inherited retinal disease. RP is a hereditary cause of blindness, which has four main modes of inheritance: Autosomal dominant RP (ADRP), autosomal recessive RP (ARRP), X-linked RP (XLRP) and dihybrid inheritance, also mitochondrial genetic and non-genetic forms. Thomas and colleagues injected rAAV2-VMD2-hMERTK vector into the sub-retinal space of RCS rats and SD rats; it showed improvement of visual function in RCS, they also performed a series of safety studies in normal SD rats, and demonstrated that no local or systemic toxicity was detected after either dose of vector delivery and no indication of vector spread outside the treated eye. This group also prompted a phase I clinical trial in which rAAV2-VMD2-hMERTK vector were injected into the sub-retinal space of patients with retinal disease due to MERTK mutations. To continue reading the paper, click here .

Qi, Yang-Fan MDa; Ma, Xiaoping MDb; Lin, Shuang-Zhu PhDc; Wang, Wan-Qi MDa; Li, Jia-Yi MDa; Chen, Qian-Dui MDa; Liu, Li PhDb, | Medicine | December 22, 2023 | 102(51) | p e36357 | DOI: 10.1097/MD.0000000000036357 Abstract Rational Retinitis pigmentosa with or without skeletal abnormalities (RPSKA) is an autosomal recessive disorder caused by mutations in the CWC27 gene. Skeletal dysplasia and non-syndromic retinitis pigmentosa are typical manifestations, and most patients present with retinopathy such as retinitis pigmentosa and limited visual field. Its clinical manifestations are complex and diverse, often involving multiple systems. Examples include short finger deformities, peculiar facial features, short stature, and neurodevelopmental abnormalities, and it is easy to misdiagnose clinically, and early diagnosis is crucial for prognosis. Patient concerns A 2-year and 2-month-old female child was admitted to the hospital due to “unsteady walking alone and slow reaction for more than half a year.” After admission, the child was found to have delayed motor development, accompanied by special face, abnormal physical examination of the nervous system, cranial MRI Dandy-Walker malformation, considering developmental delay. Diagnoses Whole exome sequencing of the family line revealed the presence of a c.617(exon7)C>A pure mutation in the CWC27 gene in the affected child (this locus has been reported in the clinical literature); the final diagnosis is RPSKA. Interventions Unfortunately, there is no specific drug for the disease; we give children rehabilitation training treatment. Outcomes During follow-up process we found that children’s condition is better than before. Lessons subsections as per style We reported a case of RPSKA caused by mutations in the CWC27 gene. This study adds to our understanding of the clinical phenotype of TBL1XR1 mutations and provides a realistic and reliable basis for clinicians. Introduction The CWC27 gene (OMIM: 617170), located on chromosome 5q12.3, is a splice-like cyclophilic peptidyl-prolyl cis-trans isomerase[ 1 ]; Phillips et al[ 2 ] first reported children with the gene mutation in 1981. Inheritance is autosomal recessive, and common variants include homozygous and complex heterozygous variants. Retinitis pigmentosa with or without skeletal anomalies (RPSKA) (OMIM: 250410) is an autosomal recessive genetic disease caused by mutations in the CWC27 gene. The disease can involve multiple systems, with skeletal dysplasia and retinopathy as the main manifestations, accompanied by developmental abnormalities. The characteristic retinopathy is manifested as retinitis pigmentosa, restricted visual field, ptosis of eyelid fissure, and thinning of retinal blood vessels.[ 3 , 4 ] We report a case of a homozygous variant of the CWC27 gene in which onset of stunting was the main manifestation. Our report enriches the understanding of the clinical phenotype of CWC27 mutations and provides a realistic and reliable basis for clinicians. Case presentation A 2-year and 2-month-old female child suffered from gross motor developmental delay after birth. She could stand upright at 2 months, turn over at 4 months, sit at 6 months, and unconsciously utter “baba, mama” at 1 year and 1 month. Sound, crawling at 1 year and 4 months, walking alone at 1 year and 8 months, still walking unsteadily, slow response, can only consciously pronounce “Dad, Mom.” During the period, the child was 8 months old because she could not climb and was treated in our hospital, and the physical examination found that the muscle tone was high, and there was no significant improvement after giving rehabilitation training guidance, and half a month ago she was treated in our hospital because of “unstable walking alone and slow response,” and was admitted to the hospital after the outpatient clinic with the main complaint of “unstable walking alone and slow response for more than half a year.” Current symptoms: the child is unstable alone, slow response, can only consciously make “dad, mother” sound, no cough and fever, no vomiting and diarrhea, diet and sleep, normal stool. After admission, we asked the children’s birth and growth history in detail; the children were G4P2, 36 + 6 weeks cesarean section, BW 2000 g (−3SD), born with little amniotic fluid, asphyxiation and rescue history, post-natal diagnosis of “respiratory distress, preterm delivery, low birth weight infants, neonatal pneumonia, neonatal hypoglycemia, ventricular, atrial, patent ductus arteriosus, hyperbilirubinemia, renal horseshoe kidney, hearing abnormalities of both ears,” after treatment were better discharged (unknown). The mother had been suffering from “upper respiratory tract infection” for more than 10 days during pregnancy, without special treatment, before birth due to “little amniotic fluid” oxygen for 2 days, and still denied the special situation. After birth, the children’s great movement development lags behind their peers, they can erect at the age of 2 months, turn over at the age of 4 months, sit at the age of 6 months, unconsciously give the sounds of “baba, mama” at the age of 1 year, climb at the age of 1 year, 4 months, walk alone at the age of 1 year, and still walk unstably, have slow reaction, have slow eye contact with people, can express sign language, but can only give the sounds of “Mom and Dad,” are happy to talk to themselves, are happy to open the door, can execute simple instructions, cannot show the inconvenience. Continue reading entire article