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Preclinical dose response study shows NR2E3 can attenuate retinal degeneration in the retinitis pigmentosa mouse model RhoP23H+/−

Jan 26, 2024

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.



 

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