53 a neuro specific gene therapy approach to treat cognitive impairment in down syndrome by rna interference

Down syndrome (DS) is a genetic disorder caused by the presence of a third copy of chromosome 21. DS affects multiple organs, resulting in characteristic facial features, muscular hypotonia, heart defects, brain development impairment, and varying degrees of intellectual disability. Trisomic mouse models of DS reproduce the main cognitive disabilities of the human syndrome. In particular, DS mice show structural and functional synaptic impairment as well as learning and memory deficits, largely determined by altered GABAergic transmission through chloride-permeable GABAa receptors (GABAaR). In particular, we have recently found that intracellular chloride accumulation shifts GABAAR-mediated signaling from inhibitory to excitatory in the adult brain of the Ts65Dn mouse model of DS. Accordingly, intracellular chloride accumulation was paralleled by increased expression of the chloride importer NKCC1 (Na-K-Cl cotransporter) in the brains of both trisomic mice and DS patients.Our findings on NKCC1 as a pivotal molecular target for the rescue of cognitive deficits in DS opens the possibility of a gene therapy approach to treat the disease. Here, to normalize NKCC1 expression and rescue synaptic dysfunctions as well as cognitive deficits in Ts65Dn mice we have developed and characterized a knock-down approach to normalize NKCC1 activity. Reducing the expression of the chloride importer NKCC1 by RNA interference restored GABAAR-mediated inhibition and also rescued the structural dendritic deficits found in trisomic neurons in vitro. Most importantly, focal administration of an AAV expressing a silencing RNA under the transcriptional control of a neuron-specific promoter in the hippocampus of Ts65Dn animals mediated NKCC1 knockdown in vivo and rescued behavioral performance on different learning and memory tests at levels undistinguishable from those of WT mice.Our findings demonstrate that NKCC1 overexpression drives excitatory GABAAR signaling in trisomic cells, leading to structural neuronal abnormalities and behavioral impairments in DS mice. Moreover, our study identifies a new gene therapy target for treatments aimed at rescuing cognitive disabilities in individuals with DS

Neurotrophic-mimetic strategy to rescue synaptic plasticity and cognitive functions in a mouse model of Down syndrome

Down syndrome (DS) or trisomy 21 is the most frequent genetic cause of intellectual disability in children and adults. Although numerous studies have shown that cognitive impairment possibly arises from dysfunction of the hippocampal circuit, there has been little progress in defining effective treatments. Previous studies have shown that impaired synaptic plasticity of mature hippocampal neurons and decreased hippocampal adult neurogenesis are main determinants in reducing cognitive functions in DS animal models. Currently, most preclinical therapeutic approaches in DS mice have focused on rescuing either one or the other of these impairments. Here, we have found that the expression of Brain-Derived Neurotrophic Factor (BDNF) is decreased in the brains of individuals with DS. Interestingly, a large body of literature indicates that BDNF signaling modulates both synaptic plasticity, and adult neurogenesis. Therefore, we propose here to promote BDNF/TrkB signaling using a BDNF-mimetic drug with the twofold aim of rescuing synaptic plasticity and increase adult neurogenesis toward the rescue of cognitive functions in the Ts65Dn mouse model of DS. Our results indicate that indeed promoting BDNF/TrkB signaling rescued hippocampal synaptic plasticity, increased hippocampal adult neurogenesis and restored cognitive performances in different behavioral tasks in Ts65Dn mice. The molecular mechanisms of impaired BDNF/TrkB signaling in trisomic mice are currently under investigation. Overall, our experiments show in a reliable animal model of DS the efficacy of a novel and multifaceted therapeutic approach with good potential to be translated into clinical practice

International consensus recommendations on the diagnostic work-up for malformations of cortical development

Malformations of cortical development (MCDs) are neurodevelopmental disorders that result from abnormal development of the cerebral cortex in utero. MCDs place a substantial burden on affected individuals, their families and societies worldwide, as these individuals can experience lifelong drug-resistant epilepsy, cerebral palsy, feeding difficulties, intellectual disability and other neurological and behavioural anomalies. The diagnostic pathway for MCDs is complex owing to wide variations in presentation and aetiology, thereby hampering timely and adequate management. In this article, the international MCD network Neuro-MIG provides consensus recommendations to aid both expert and non-expert clinicians in the diagnostic work-up of MCDs with the aim of improving patient management worldwide. We reviewed the literature on clinical presentation, aetiology and diagnostic approaches for the main MCD subtypes and collected data on current practices and recommendations from clinicians and diagnostic laboratories within Neuro-MIG. We reached consensus by 42 professionals from 20 countries, using expert discussions and a Delphi consensus process. We present a diagnostic workflow that can be applied to any individual with MCD and a comprehensive list of MCD-related genes with their associated phenotypes. The workflow is designed to maximize the diagnostic yield and increase the number of patients receiving personalized care and counselling on prognosis and recurrence risk

Gene editing in photoreceptor progenitors prevents visual function loss in a mouse model of retinal degeneration

Purpose:Currently, there is no known cure for Retinitis pigmentosa (RP). Even if some treatments can slow down the progression of the disease, none of them can effectively stop retinal degeneration. We exploited the possibility of an early intervention in photoreceptor progenitors aiming at preventing cell death. For our purpose, we selected the Rd10 mouse model, which carries a point mutation in a gene associated with human RP. We designed a CRISPR/Cas9 gene editing system to repair the mutation taking advantage of the increased activity of the homologous directed repair mechanism in dividing cells. Methods:The efficiency of the editing system (composed of guide RNA, Cas9, and DNA repair template) was first tested in vitro in neural progenitor cells (NPCs) derived from Rd10 mice (n=3). The constructs were then injected in vivo in the subretinal space of Rd10 pups at postnatal day (P) 1.5 (single treated, ST) or at P1.5 and P8 (multiple treated, MT). One eye was injected, while the other one was kept as internal control. The injection was followed by electroporation (electric field: 40 V/cm). The visual acuity was measured at P28 with the optomotor test in ST (n=43), MT (n=12), sham (n=20), untreated Rd10 (NT, n=13), and WT (n=18) mice. One tailed Student’s t-test was used to compare control and treated eyes, while one-way ANOVA was used to compare different groups. Moreover, the flash visually evoked potentials (fVEPs) were recorded from the visual cortex of ST (n=19), MT(n=12), sham (n=17), NT (n=4), and WT(n=10) mice at P33. Results:The net efficiency of the CRISPR/Cas9-mediated DNA editing in NPCs was 52.8±11.1%. The visual acuity in the treated eye was significantly higher compared to the control eye in ST (0.22±0.02c/d vs 0.12±0.01c/d, p<0.01) and MT mice (0.27±0.02c/d vs 0.15±0.02c/d, p<0.01). ST and MT mice had a significantly higher visual acuity compared to sham (0.11±0.01c/d, p<0.01) and NT mice (0.11±0.07c/d, p<0.01). Preliminary measurements of the fVEPs showed a partial recovery of the light-evoked response in MT mice. Conclusions:Our results strongly suggest a positive effect of the CRISPR/Cas9-based therapy on photoreceptor survival in our model of RP. However, additional morphological analyses and electrophysiological tests at different time points are needed to assess the preservation of the retinal outer nuclear layer and the functionality of the visual pathways

Preventing visual function loss in the rd10 mouse model of retinitis pigmentosa using gene editing

Currently, there is no known cure for retinitis pigmentosa (RP). Even if some treatments can slow down the progression of the disease, none of them can effectively stop retinal degeneration. This study exploits the possibility of an early intervention in photoreceptor progenitors aiming at preventing cell death. For this purpose, we selected the rd10 mouse model, which carries a point mutation in a gene associated with human RP. We designed a CRISPR/Cas9 gene editing system to repair the mutation taking advantage of the increased activity of the homologous directed repair mechanism in dividing cells. The efficiency of the editing system (composed of guide RNA, Cas9, and DNA repair template) was first tested in vitro in neural progenitor cells derived from rd10 mice (52.8±11.1%, n=3). The constructs were then injected in vivo in the subretinal space of rd10 pups either at postnatal day (P) 3 (early treated, ET) or at P8 (late treated, LT); we also tried a P3-P8 combined treatment (multiple treated, MT). One eye was injected, while the other one was kept as internal control. The injection was followed by electroporation (electric field: 40 V/cm). Histological analysis of the eyes showed GFP expression in the photoreceptors layer starting from 2 days after electroporation. The visual acuity was measured at P30, P60 and P90 with the optomotor response test in ET, MT, LT, sham treated (ST), non-treated rd10 (NT), and WT (WT) mice. The treated eye showed a higher visual acuity than the control eye in ET, MT and LT for all the time points tested, despite a decreased visual acuity at P90 (p<0.01, one-tailed Student’s t-test). ET, MT, and LT mice had a significantly higher visual acuity compared to ST and NT mice for all the time points tested (p<0.01, one-way ANOVA + Tukey). Moreover, in order to test the integrity of the cortical visual pathway, the flash visually evoked potentials (fVEPs) were recorded from the visual cortex of ET, MT, LT, ST, NT, and WT mice at P90. We observed a partial recovery of the light-evoked response in the visual cortex of ET and LT mice (n.s and p<0.05; one-way ANOVA + Tukey), compared to ST and NT mice. Our results strongly suggest a positive effect of the CRISPR/Cas9-based therapy on photoreceptors survival in our model of RP. In the future, we would like to perform additional morphological analyses to better understand the correlation between the injection site in the retina and the specificity of the targeted visual circuits

Neurotrophic-mimetic strategy to rescue synaptic plasticity and cognitive functions in a mouse model of Down syndrome

Down syndrome (DS) is caused by the triplication of human chromosome 21, and it is the most frequent genetic cause of cognitive disabilities. Although numerous studies have shown that cognitive impairment possibly arises from dysfunction of the hippocampal circuit, there is little insight into neurobiological bases of these abnormalities, and thus, there has been little progress in defining effective treatments. The trisomic Ts65Dn mouse model of DS reproduces the essential cognitive disabilities of the human syndrome. Previous studies in this model have shown that impaired synaptic plasticity of mature hippocampal neurons and decreased hippocampal adult neurogenesis are main determinants in reducing cognitive functions in DS animal models. Currently, most preclinical therapeutic approaches in the DS mouse models have focused on rescuing either one or the other of these impairments. Interestingly, we have found that the expression of Brain-Derived Neurotrophic Factor (BDNF) is decreased in the brains of DS patients. On the other hand, BDNF signaling modulates both synaptic plasticity, and adult neurogenesis. Therefore, we propose to promote BDNF/TrkB signaling using a BDNF-mimetic drug with the twofold aim of rescuing synaptic plasticity and increase adult neurogenesis toward the rescue of cognitive functions in Ts65Dn mice. Our results indicate that indeed promoting BDNF/TrkB signaling rescued hippocampal synaptic plasticity, increased dentate adult neurogenesis and restored cognitive performances in different behavioral tasks in Ts65Dn mice. Overall, our experiments show in a reliable animal model of DS the efficacy of a novel and multifaceted therapeutic approach with good potential to be translated into clinical practice

Physical exercise rescues adult neurogenesis, synaptic plasticity and memory in down syndrome mice

Down syndrome (DS), caused by the triplication of human chromosome 21, is the most frequent genetic cause of mental retardation. The Ts65Dn mouse model of DS show many neurological similarities to the human syndrome, including decreased hippocampal neurogenesis and cognitive impairment. In this study, we have investigated the effect of aerobic physical exercise on adult neurogenesis, synaptic plasticity and memory in Ts65Dn mice. Exposure of adult Ts65Dn mice to running wheels for one month increased the proliferation of neuronal precursor cell and stimulated adult neurogenesis in the hippocampal dentate gyrus. Moreover, physical exercise promoted the recovery of hippocampal synaptic plasticity and, most importantly, fully restored learning and memory in different behavioral task in trisomic mice. These findings demonstrate that Ts65Dn mice benefit from voluntary wheel running and provide evidence that physical exercise could represent a valuable complementary therapy for pharmacological interventions aimed at rescuing cognitive disabilities in DS patients

Aerobic exercise and a BDNF-mimetic therapy rescue learning and memory in a mouse model of Down syndrome

Abstract Down syndrome (DS) is caused by the triplication of human chromosome 21 and represents the most frequent genetic cause of intellectual disability. The trisomic Ts65Dn mouse model of DS shows synaptic deficits and reproduces the essential cognitive disabilities of the human syndrome. Aerobic exercise improved various neurophysiological dysfunctions in Ts65Dn mice, including hippocampal synaptic deficits, by promoting synaptogenesis and neurotransmission at glutamatergic terminals. Most importantly, the same intervention also prompted the recovery of hippocampal adult neurogenesis and synaptic plasticity and restored cognitive performance in trisomic mice. Additionally, the expression of brain-derived neurotrophic factor (BDNF) was markedly decreased in the hippocampus of patients with DS. Since the positive effect of exercise was paralleled by increased BDNF expression in trisomic mice, we investigated the effectiveness of a BDNF-mimetic treatment with 7,8-dihydroxyflavone at alleviating intellectual disabilities in the DS model. Pharmacological stimulation of BDNF signaling rescued synaptic plasticity and memory deficits in Ts65Dn mice. Based on our findings, Ts65Dn mice benefit from interventions aimed at promoting brain plasticity, and we provide evidence that BDNF signaling represents a potentially new pharmacological target for treatments aimed at rescuing cognitive disabilities in patients with DS

Gene Editing Preserves Visual Functions in a Mouse Model of Retinal Degeneration

Inherited retinal dystrophies (IRDs) are a large and heterogeneous group of degenerative diseases caused by mutations in various genes. Given the favorable anatomical and immunological characteristics of the eye, gene therapy holds great potential for their treatment. Our goal is to validate the preservation of visual functions by viral-free homology directed repair (HDR) in an autosomal recessive loss of function mutation. We used a tailored gene editing system based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to prevent retinal photoreceptor death in the retinal degeneration 10 (Rd10) mouse model of retinitis pigmentosa. We tested the gene editing tool in vitro and then used in vivo subretinal electroporation to deliver it to one of the retinas of mouse pups at different stages of photoreceptor differentiation. Three months after gene editing, the treated eye exhibited a higher visual acuity compared to the untreated eye. Moreover, we observed preservation of light-evoked responses both in explanted retinas and in the visual cortex of treated animals. Our study validates a CRISPR/Cas9-based therapy as a valuable new approach for the treatment of retinitis pigmentosa caused by autosomal recessive loss-of-function point mutations