Effect of Allele-Specific Clcn7G213R siRNA Delivered Via a Novel Nanocarrier on Bone Phenotypes in ADO2 Mice on 129S Background.

Autosomal dominant osteopetrosis type 2 (ADO2) is a rare inherited bone disorder characterised by dense but brittle bones. It displays striking phenotypic variability, with the most severe symptoms, including blindness and bone marrow failure. Disease management largely relies on symptomatic treatment since there is no safe and effective treatment. Most ADO2 cases are caused by heterozygous loss-of-function mutations in the CLCN7 gene, which encodes an essential Cl-/H+ antiporter for proper bone resorption by osteoclasts. Thus, siRNA-mediated silencing of the mutant allele is a promising therapeutic approach, but targeting bone for first-in-human translation remains challenging. Here, we demonstrate the utility of silicon-stabilised hybrid lipid nanoparticles (sshLNPs) as a next-generation nucleic acid nanocarrier capable of delivering allele-specific siRNA to bone. Using a Clcn7G213R knock-in mouse model recapitulating one of the most common human ADO2 mutations and based on the 129S genetic background (which produces the most severe disease phenotype amongst current models), we show substantial knockdown of the mutant allele in femur when siRNA targeting the pathogenic variant is delivered by sshLNPs. We observed lower areal bone mineral density in femur and reduced trabecular thickness in femur and tibia, when siRNA-loaded sshLNPs were administered subcutaneously (representing the most relevant administration route for clinical adoption and patient adherence). Importantly, sshLNPs have improved stability over conventional LNPs and enable 'post hoc loading' for point-of-care formulation. The treatment was well tolerated, suggesting that sshLNP-enabled gene therapy might allow successful clinical translation of essential new treatments for ADO2 and potentially other rare genetic bone diseases.

Novel hybrid silicon-lipid nanoparticles deliver a siRNA to cure autosomal dominant osteopetrosis in mice. Implications for gene therapy in humans.

Rare skeletal diseases are still in need of proper clinically available transfection agents as the major challenge for first-in-human translation relates to intrinsic difficulty in targeting bone without exacerbating any inherent toxicity due to used vector. SiSaf's silicon stabilized hybrid lipid nanoparticles (sshLNPs) constitute next-generation non-viral vectors able to retain the integrity and stability of constructs and to accommodate considerable payloads of biologicals, without requiring cold-chain storage. sshLNP was complexed with a small interfering RNA (siRNA) specifically designed against the human CLCN7G215R mRNA. When tested via single intraperitoneal injection in pre-puberal autosomal dominant osteopetrosis type 2 (ADO2) mice, carrying a heterozygous mutation of the Clcn7 gene (Clcn7G213R), sshLNP, this significantly downregulated the Clcn7G213R related mRNA levels in femurs at 48 h. Confirmatory results were observed at 2 weeks and 4 weeks after treatments (3 intraperitoneal injections/week), with rescue of the bone phenotype and demonstrating safety. The pre-clinical results will enable advanced preclinical development of RNA-based therapy for orphan and genetic skeletal disorders by safely and effectively delivering biologicals of interest to cure human systemic conditions.

Delivery of siRNA to the Eye: Protocol for a Feasibility Study to Assess Novel Delivery System for Topical Delivery of siRNA Therapeutics to the Ocular Surface.

Drug delivery to the eye remains a real challenge due to the presence of ocular anatomical barriers and physiological protective mechanisms. The lack of effective siRNA delivery mechanism has hampered the real potential of RNAi therapy, but recent literature suggests that nanocarrier systems show great promise in enhancing siRNA bioavailability and reducing the need for repeated intraocular injections. A diverse range of materials are under exploration worldwide, including natural and synthetic polymers, liposomes, peptides, and dendrimeric nanomaterials. This chapter describes a simple workflow for feasibility assessment of a proposed ocular surface siRNA delivery system. Gel retardation assay is used for investigation of optimal siRNA to carrier loading ratio. Fluorescent siRNA allows for initial in vitro testing of cellular uptake to corneal epithelial cells and investigation of in vivo siRNA delivery into mouse cornea by live animal imaging and fluorescence microscopy.

Topical siRNA delivery to the cornea and anterior eye by hybrid silicon-lipid nanoparticles.

The major unmet need and crucial challenge hampering the exciting potential of RNAi therapeutics in ophthalmology is to find an effective, safe and non-invasive means of delivering siRNA to the cornea. Although all tissues of the eye are accessible by injection, topical application is preferable for the frequent treatment regimen that would be necessary for siRNA-induced gene silencing. However, the ocular surface is one of the more complex biological barriers for drug delivery due to the combined effect of short contact time, tear dilution and poor corneal cell penetration. Using nanotechnology to overcome the challenges, we developed a unique silicon-based delivery platform for ocular delivery of siRNA. This biocompatible hybrid of porous silicon nanoparticles and lipids has demonstrated an ability to bind nucleic acid and deliver functional siRNA to corneal cells both in vitro and in vivo. Potent transfection of human corneal epithelial cells with siRNA-ProSilic® formulation was followed by a successful downregulation of reporter protein expression. Moreover, siRNA complexed with this silicon-based hybrid and applied in vivo topically to mice eyes penetrated across all cornea layers and resulted in a significant reduction of the targeted protein expression in corneal epithelium. In terms of siRNA loading capacity, system versatility, and potency of action, ProSilic provides unique attributes as a biodegradable delivery platform for therapeutic oligonucleotides.

Chronic social isolation in adaptation of HPA axis to heterotypic stress.

BACKGROUND: Social crowding and isolation are recognized as major stressors and risk factors for development of psychiatric disorders. Chronic isolation stress (IS) and crowding stress (CS) activate neuroendocrine and neurochemical mechanisms, that activate the hypothalamic-pituitary-adrenal (HPA) axis. Changes of the plasma level of interleukin-1ß (IL-1ß), ACTH and corticosterone (CORT) after chronic psychosocial IS and CS were investigated. METHODS: Control rats were kept 5 per cage and not stressed. Stressed groups were subjected to either CS for 3, 7, 14days+restraint stress (RS) or IS for (11days) before this treatment was applied. Crowded rats were remained (24 in one cage) and RS rats were restrained for 10min. Total CORT, ACTH and IL-1ß levels were measured using commercially available kits. RESULTS: Social CS for 3days significantly increased plasma IL-1ß level. Social IS increased plasma IL-1ß level after longer period of subsequent CS 7 and 14days, than ACTH and CORT, after 3 and 7days. Prior IS significantly increased plasma IL-1ß level induced by subsequent combined CS for 3days+acute RS, but significantly or totally inhibited the acute stress-induced increase of plasma IL-1ß level after 7 and 14days of combined stress. IS, by contrast, strongly inhibited the increase of plasma ACTH and CORT level induced by combined CS+acute RS. CONCLUSION: Chronic IS augments the changes of IL-1ß level induced by a longer crowding period than ACTH and CORT. Modulatory action of IL-1ß and pituitary-adrenocortical hormones adaptation to chronic social stress is asynchronous.

Psychosocial stress inhibits additional stress-induced hyperexpression of NO synthases and IL-1ß in brain structures.

BACKGROUND: The aim of this study was to compare the expression of interleukin-1ß (IL-1ß), neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in the prefrontal cortex (PFC), hippocampus (HIP) and hypothalamus (HT) during chronic crowding (CS) (psychosocial) and restraint (RS) (physico-psychological) stress. Adaptational changes of these stress mediators to a subsequent acute RS, in two models of chronic stress were investigated. METHODS: Rats were crowded (24 in one cage) or restrained in metal tubes for 10min twice a day for 3, 7, and 14 consecutive days and decapitated. For determination of adaptational changes the chronically crowded and restrained rats 24h after the last stress session were subjected to a single 10min RS. The IL-1ß, nNOS and iNOS protein levels in brain structures samples were analyzed by Western blot procedure. RESULTS: Chronic CS for 3days did not markedly change the subsequent acute stress induced expression of nNOS, iNOS and IL-1ß protein level in PFC and iNOS protein level in HT. CS markedly decreased the expression of nNOS, iNOS and IL-1ß in HIP. By contrast, parallel chronic RS, significantly increased the subsequent acute stress-induced expression of iNOS and IL-1ß in PFC and considerably increased iNOS level in HT. CONCLUSION: Chronic psychosocial stress, may protect against possible harmful action of hyperproduction of iNOS and iNOS derived nitric oxide (NO) mainly in PFC and HIP. By contrast, chronic physico-psychosocial stress may strongly potentiate additional stress-induced harmful effects of NOS and IL-1ß hyperproduction.

Influence of chronic stress on brain corticosteroid receptors and HPA axis activity.

BACKGROUND: Disruption of the glucocorticoid negative feedback system evoked in animals by chronic stress can be induced by downregulation of glucocorticoid receptors (GRs) in several brain regions. In the present study, the dynamics of the changes in GRs, in brain structures involved in stress reactions, prefrontal cortex, hippocampus and hypothalamus was compared with the peripheral hypothalamo-pituitary-adrenocortical (HPA) axis hormones response to chronic stress. METHODS: Rats were exposed to 10 min restraint or restrained twice a day for 3, 7 or 14 days, and 24 h after the last stress session exposed to homotypic stress for 10 min. Control rats were not restrained. After rapid decapitation at 0, 1, 2, and 3 h after stress termination, trunk blood for plasma adrenocorticotropic hormone (ACTH) and corticosterone determinations was collected and prefrontal cortex, hippocampus and hypothalamus were excised and frozen. Plasma hormones were determined using commercially available kits and glucocorticoids and mineralocorticoids protein levels in brain structure samples were determined by western blot procedure. RESULTS: Restraint stress alone significantly decreased glucocorticoid receptor (GR) level in prefrontal cortex and hippocampus, and increased mineralocorticoid receptor (MR) level in hypothalamus. Prior repeated stress for 3 days significantly increased GR protein level in hippocampus and diminished that level in hypothalamus in 7 days stressed rats. Acute stress-induced strong increase in plasma ACTH and corticosterone levels decreased to control level after 1 or 2 h, respectively. Prior repeated stress for 3 days markedly diminished the fall in plasma ACTH level and repeated stress for 7 days moderately deepened this decrease. Plasma ACTH level induced by homotypic stress in rats exposed to restraint for 3, 7, and 14 days did not markedly differ from its control level, whereas plasma corticosterone response was significantly diminished. The fast decrease of stress-induced high plasma ACTH and corticosterone levels was accompanied by a parallel decline of GR level only in prefrontal cortex but not in the hippocampus or hypothalamus. CONCLUSIONS: Comparison of the dynamics of changes in plasma ACTH and corticosterone level with respective alterations in GR and MR in brain structures suggests that the buffering effect of repeated stress depends on the period of habituation to stress and the brain structure involved in regulation of these stress response.

Cytokines, prostaglandins and nitric oxide in the regulation of stress-response systems.

Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis is accepted as one of the fundamental biological mechanisms that underlie major depression. This hyperactivity is caused by diminished feedback inhibition of glucocorticoid (GC)-induced reduction of HPA axis signaling and increased corticotrophin-releasing hormone (CRH) secretion from the hypothalamic paraventricular nucleus (PVN) and extra-hypothalamic neurons. During chronic stress-induced inhibition of systemic feedback, cytosolic glucocorticoid receptor (GR) levels were significantly changed in the prefrontal cortex (PFC) and hippocampus, both structures known to be deeply involved in the pathogenesis of depression. Cytokines secreted by both immune and non-immune cells can markedly affect neurotransmission within regulatory brain circuits related to the expression of emotions; cytokines may also induce hormonal changes similar to those observed following exposure to stress. Proinflammatory cytokines, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are implicated in the etiologies of clinical depression and anxiety disorders. Prolonged stress responses and cytokines impair neuronal plasticity and stimulation of neurotransmission. Exposure to acute stress and IL-1ß markedly increased IL-1ß levels in the PFC, hippocampus and hypothalamus, as well as overall HPA axis activity. Repeated stress sensitized the HPA axis response to IL-1ß. Inflammatory responses in the brain contribute to cellular damage associated with neuropsychiatric diseases related to stress. Physical, psychological or combined-stress conditions evoke a proinflammatory response in the brain and other systems, characterized by a complex release of several inflammatory mediators including cytokines, prostanoids, nitric oxide (NO) and transcription factors. Induced CRH release involves IL-1, IL-6 and TNF-α, for stimulation adrenocorticotropic hormone (ACTH) release from the anterior pituitary. NO also participates in signal transduction pathways that result in the release of corticosterone from the adrenal gland. NO participates in multiple interactions between neuroendocrine and neuroimmune systems in physiological and pathological processes. Neuronal NO synthase (nNOS) modulates learning and memory and is involved in development of neuropsychiatric diseases, including depression. Nitric oxide generated in response to stress exposure is associated with depression-like and anxiety-like behaviors. In the central nervous system (CNS), prostaglandins (PG) generated by the cyclooxygenase (COX) enzyme are involved in the regulation of HPA axis activity. Prior exposure to chronic stress alters constitutive (COX-1) and inducible (COX-2) cyclooxygenase responses to homotypic stress differently in the PFC, hippocampus and hypothalamus. Both PG and NO generated within the PVN participate in this modulation. Acute stress affects the functionality of COX/PG and NOS/NO systems in brain structures. The complex responses of central and peripheral pathways to acute and chronic stress involve cytokines, NO and PG systems that regulate and turn off responses that would be potentially harmful for cellular homeostasis and overall health.

Effect of repeated restraint on homotypic stress-induced nitric oxide synthases expression in brain structures regulating HPA axis.

BACKGROUND: Restraint stress (RS) markedly increases interleukin 1-ß (IL-1ß) generation in brain structures involved in hypothalamic-pituitary adrenocortical (HPA) axis regulation. The IL-1ß-induced transient stimulation of HPA axis activity was parallel in time and magnitude to respective changes in regulation of HPA activity. In the present experiment the expression of neuron al and inducible nitric oxide synthase (nNOS and iNOS) were investigated in prefrontal cortex, hippocampus and hypothalamus in response to acute restraint stress in control and prior repeatedly restrained rats. METHODS: Experiments were performed on male Wistar rats which were exposed to 10 min restraint stress or restrained twice a day for 3 days, and 24 h after the last stress period exposed to homotypic stress for 10 min. After rapid decapitation at 0, 1, 2 and 3 h after cessation of stress, trunk blood was collected and prefrontal cortex, hippocampus and hypothalamus were excised and frozen. Interleukin-1ß, adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels were determined in plasma using commercially available kits and neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in brain structure samples were analyzed by western blot procedure. RESULTS: Prior repeated restraint stress enhanced the acute restraint stress induced increase in IL-1ß levels in all three structures examined. Restraint stress for 10 min moderately decreased nNOS level in prefrontal cortex in control rats, augmented this level in hippocampus and markedly increased nNOS level in hypothalamus. Restraint itself significantly decreased iNOS level in prefrontal cortex, while it enhanced iNOS level in hippocampus and hypothalamus. Prior restraint stress for 3 days enhanced the nNOS level in prefrontal cortex and hippocampus and did not substantially affect nNOS levels response in hypothalamus. Repeated restraint stress considerably augmented the iNOS levels in both prefrontal cortex, hippocampus and hypothalamus induced by followed homotypic stress. CONCLUSION: These results indicate that during restraint stress nNOS regulate formation of low amount of NO and the high-output generation of NO is effected by inducible isoform of nitric oxide synthase. Prior repeated stress significantly enhances the homotypic stress-induced nNOS and iNOS responses.

Effect of co-treatment with fluoxetine or mirtazapine and risperidone on the active behaviors and plasma corticosterone concentration in rats subjected to the forced swim test.

BACKGROUND: Several clinical reports have postulated a beneficial effect of the addition of a low dose of risperidone to the ongoing treatment with antidepressants in treatment-resistant depression. METHODS: The present study aimed to examine the effect of treatment with fluoxetine or mirtazapine, given separately or jointly with risperidone, on active behavior and plasma corticosterone level in male Wistar rats subjected to the forced swim test (FST). RESULTS: The obtained results showed that fluoxetine (5 mg/kg), mirtazapine (5 and 10 mg/kg) or risperidone (0.05 and 0.1 mg/kg) did not change the active behavior of rats in the FST. However, co-treatment with fluoxetine (10 mg/kg) and risperidone (0.1 mg/kg) induced an antidepressant-like effect in that test because it significantly increased the swimming time and decreased the immobility time, while combined treatment with mirtazapine at 5 and 10 mg/kg and risperidone at 0.05 and 0.1 mg/kg evoked a significant increase in the swimming time and also climbing, and decreased the immobility time. WAY 100635 (a 5-HT(1A) receptor antagonist) at a dose of 0.1 mg/kg inhibited the antidepressant-like effect induced by co-administration of fluoxetine or mirtazapine and risperidone. Active behavior in that test did not reflect an increase in general activity, since combined treatment with fluoxetine or mirtazapine and risperidone failed to enhance the exploratory activity of rats. Co-treatment with fluoxetine or mirtazapine and risperidone did not reduce the stress-induced increase in plasma corticosterone concentration in animals subjected to the FST. CONCLUSION: The obtained results indicate that risperidone applied in a low dose enhances the antidepressant-like activity of fluoxetine and mirtazapine in the FST (but does not normalize the stress-induced increase in corticosterone level in these rats), and that 5-HT(1A) receptors may play some role in these effects.

Brain nitric oxide synthases in the interleukin-1ß-induced activation of hypothalamic-pituitary-adrenal axis.

BACKGROUND: Interleukin-1ß (IL-1ß), the major cytokine involved in activation of hypothalamic-pituitary-adrenal (HPA) axis modulates both central and peripheral components regulating HPA activity. The role of nitric oxide (NO) generated by neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in brain structures involved in HPA axis regulation has not been elucidated. The aim of the study was to assess the receptor selectivity of IL-1ß stimulatory action on HPA axis and to determine the involvement of nNOS and iNOS in this stimulation. METHODS: Experiments were performed on male Wistar rats which were injected intraperitoneally (ip) with IL-1ß (5 µg/kg) or IL-1 receptor antagonist (IL-1ra) (50 µg/kg or 100 µg/kg) 15 min before IL-1ß. Rats were sacrificed by rapid decapitation 1, 2 or 3 h after IL-1ß administration. Trunk blood for ACTH, corticosterone and IL-1ß determinations was collected and prefrontal cortex, hippocampus and hypothalamus were excised and snap frozen. Western blot analyses were performed and IL-1ß, nNOS and iNOS protein were determined in brain structures samples. RESULTS: IL-1ß significantly increased plasma ACTH, corticosterone and IL-1ß levels during 2 h after ip administration. IL-1 receptor antagonist was able to abolish the stimulatory effect of IL-1ß on plasma ACTH and corticosterone levels and significantly, but not totally, reduced plasma IL-1ß level. The role of NO in prefrontal cortex, hippocampus and hypothalamus in the IL-1ß-induced HPA axis activity alterations was determined by measuring the changes in nNOS and iNOS levels. The highest level of both izoenzymes 1 h following IL-1ß administration decreased in a regular, parallel manner 2 and 3 h later, approaching control values. These changes were almost totally prevented by pretreatment with IL-1 receptor antagonist. In the hypothalamus the IL-1ß-induced initial significant increase of nNOS regularly decreased in a modest rate and remained at significant higher level compared to control values. By contrast, iNOS level gradually increased 2 and 3 h after IL-1ß administration in a significant time-dependent manner. The changes in both NOS izoenzyme levels in hypothalamus were suppressed by pretreatment with IL-1 receptor antagonist. Results also show that a regular and parallel decrease of nNOS in the hypothalamus and prefrontal cortex are parallel in time and magnitude to respective fall in plasma IL-1ß and ACTH levels. CONCLUSION: The present study suggests that the IL-1ß-induced transient stimulation of HPA axis activity is parallel in time and magnitude to the respective changes of nNOS in hypothalamus and prefrontal cortex, the brain structures involved in regulation of HPA axis activity.

Effect of prior stress on interleukin-1ß and HPA axis responses to acute stress.

Interleukin-1ß (IL-1ß) level is modulated during multiple stress reactions both in brain structures involved in hypothalamic-pituitary-adrenal (HPA) axis regulation and peripheral systems. Multiple distinct stressors induce different IL-1ß and HPA axis responses. The purpose of the present study was to determine if the effect of prior repeated restraint stress on IL-1ß levels in prefrontal cortex, hippocampus, hypothalamus and plasma may have an impact on alterations induced in HPA axis responses. Experiments were performed on male Wistar rats which were exposed to 10 min restraint stress twice a day for 3 days. Twenty four hours after the last stress period rats were restrained for 10 min and decapitated at 0, 1, 2 or 3 h after cessation of stress. Control rats were injected ip with saline and some of experimental groups with IL-1ß receptor antagonist (IL-1ra). After rapid decapitation, trunk blood was collected and prefrontal cortex, hippocampus and hypothalamus were excised and frozen. Interleukin-1ß, adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels were determined in plasma using commercially available kits and IL-1ß levels in brain structures samples were analyzed by western blot procedure. Repeated restraint for 3 days alone did not alter resting plasma levels of IL-1ß, and moderately augmented plasma ACTH and CORT levels and IL-1ß content in brain structures 24 h after the last restraint. IL-1ß antagonist abolished the increase in plasma levels of IL-1ß, ACTH and CORT as well as IL-1ß in brain structures in response to repeated stress and also reduced these changes induced by 10 min stress. This suggests the selectivity of IL-1ß receptors in central and peripheral mechanisms modulating the stress-induced HPA axis responses. These results indicate that repeated stress markedly increases IL-1ß production in brain structures involved in HPA axis regulation. The present results support the role of brain and peripheral IL-1ß in adaptation of HPA response during prolonged stress.