Antisense Oligonucleotide- and CRISPR-Cas9-Mediated Rescue of mRNA Splicing for a Deep Intronic CLRN1 Mutation

Mutations in CLRN1 cause Usher syndrome (USH) type III (USH3A), a disease characterized by progressive hearing impairment, retinitis pigmentosa, and vestibular dysfunction. Due to the lack of appropriate disease models, no efficient therapy for retinitis pigmentosa in USH patients exists so far. In addition, given the yet undefined functional role and expression of the different CLRN1 splice isoforms in the retina, non-causative therapies such as gene supplementation are unsuitable at this stage. In this study, we focused on the recently identified deep intronic c.254-649T>G CLRN1 splicing mutation and aimed to establish two causative treatment approaches: CRISPR-Cas9-mediated excision of the mutated intronic region and antisense oligonucleotide (AON)-mediated correction of mRNA splicing. The therapeutic potential of these approaches was validated in different cell types transiently or stably expressing CLRN1 minigenes. Both approaches led to substantial correction of the splice defect. Surprisingly, however, no synergistic effect was detected when combining both methods. Finally, the injection of naked AONs into mice expressing the mutant CLRN1 minigene in the retina also led to a significant splice rescue. We propose that both AONs and CRISPR-Cas9 are suitable strategies to initiate advanced preclinical studies for treatment of USH3A patients

Retina regeneration

Our sense of vision largely defines who we are as individuals and as a society, by shaping our perception and allowing us to communicate beyond language barriers. Blinding diseases such as inherited retinal dystrophies therefore have severe mental and socio-economic consequences, which sparked multiple efforts to halt and reverse degeneration in these patients. This thesis set out to advance different therapeutic strategies that are both clinically established or under investigation, for patients at all disease stages. To address the needs of patients with early- to mid-stage retinal degeneration, two potent AAV capsids were engineered to deliver genetic cargo via a less invasive administration route, namely intravitreal injection. The properties of these novel vectors were verified in mouse, dog, non-human primate and human retina, showing a widespread transduction of photoreceptors across species. To demonstrate the potential of these vectors in gene supplementation therapy, a mouse model of achromatopsia was treated, resulting in the rescue of cone photoreceptor function and showing signs of tissue rejuvenation. For patients with late-stage retinal degeneration, two therapeutic approaches were explored that would either confer light-sensing abilities to the remaining retinal network or replenish the missing neuronal population by reprogramming retinal glia. Regarding the former, a universal method for isolating ON bipolar cells was established that would enable the development of novel vectors for delivering optogenetic tools in a cell-specific manner. Regarding the latter, a series of exploratory experiments were performed showing the potential of pioneering factors to directly reprogram retinal microglia into neurons, highlighting the potential of regenerating the retina from within. This body of work was able to optimise existing strategies to treat inherited retinal dystrophies and offered new solutions for patients with so far limited therapeutic options

A Bioengineered In Vitro Model to Assess AAV-Based Gene Therapies for Cyclic GMP-Related Disorders

The emergence of efficient viral vectors derived from adeno-associated viruses (AAV) has led many groups to develop gene therapies for inherited monogenic diseases, such as retinal dystrophies. To evaluate the potency of new gene therapy vectors in a preclinical context, it is common to use animal models, such as gene-deficient or mutant animal models of a given human disease, and then assess vision restoration with functional or behavioral assays. While such animal models are invaluable to the preclinical testing process, they cannot be readily used as batch release tests during manufacturing or to validate biological activity at later stages of development. There is therefore a need for rapid and reliable in vitro models that can determine whether therapeutic vectors have delivered their cargo gene, and more importantly, whether this has resulted in the intended biological activity. Given our previous experience, we chose CNGA3-linked achromatopsia to develop a cell-based system to verify biological activity of AAV vectors designed to deliver a healthy CNGA3 gene copy into human cone photoreceptors. Our system is based on an immortalized cell line with high susceptibility to AAV transduction, i.e., HeLa cells, which we engineered to express a fungal rhodopsin guanylyl cyclase (RhGC) from Blastocladiella emersonii and a sensitive genetically encoded calcium indicator (GECI) under the control of a tetracycline operator. Using this system, we were able to confirm and quantify the function of the ion channel encoded by AAV/CNGA3 and differentiate between AAV vector potencies with a simple fluorometric assay. Finally, we show that this approach can be readily adapted for the assessment of phosphodiesterase function

Elements of the niche for adult stem cell expansion

Adult stem cells are crucial for tissue homeostasis. These cells reside within exclusive locations in tissues, termed niches, which protect adult stem cell fidelity and regulate their many functions through biophysical-, biochemical- and cellular-mediated mechanisms. There is a growing understanding of how these mechanisms and their components contribute towards maintaining stem cell quiescence, self-renewal, expansion and differentiation patterns. In vitro expansion of adult stem cells is a powerful tool for understanding stem cell biology, and for tissue engineering and regenerative medicine applications. However, it is technically challenging, since adult stem cell removal from their native microenvironment has negative repercussions on their sustainability. In this review, we overview specific elements of the biomimetic niche and how recreating such elements can help in vitro propagation of adult stem cells

Fumaric Acids Directly Influence Gene Expression of Neuroprotective Factors in Rodent Microglia

Dimethylfumarate (DMF) has been approved the for treatment of relapsing-remitting multiple sclerosis. The mode of action of DMF and its assumed active primary metabolite monomethylfumarate (MMF) is still not fully understood, notably for brain resident cells. Therefore we investigated potential direct effects of DMF and MMF on microglia and indirect effects on oligodendrocytes. Primary rat microglia were differentiated into M1-like, M2-like and M0 phenotypes and treated in vitro with DMF or MMF. The gene expression of pro-inflammatory and anti-inflammatory factors such as growth factors (IGF-1), interleukins (IL-10, IL-1β), chemokines (CCl3, CXCL-10) as well as cytokines (TGF-1β, TNFα), iNOS, and the mannose receptor (MRC1) was examined by determining their transcription level with qPCR, and on the protein level by ELISA and FACS analysis. Furthermore, microglia function was determined by phagocytosis assays and indirect effects on oligodendroglial proliferation and differentiation. DMF treatment of M0 and M1-like polarized microglia demonstrated an upregulation of gene expression for IGF-1 and MRC1, but not on the protein level. While the phagocytic activity remained unchanged, DMF and MMF treated microglia supernatants led to an enhanced proliferation of oligodendrocyte precursor cells (OPC). These results suggest that DMF has anti-inflammatory effects on microglia which may result in enhanced proliferation of OPC

Profiling carotenoid and phenolic compounds in fresh and canned fruit of peach cultivars: Impact of genotype and canning on their concentration

Peach (Prunus persica (L.) Batsch) is widely consumed both fresh and processed. Scarce information exists regarding the fate of phytochemicals after the peach canning process. The aim of the current study was to evaluate the compositional variability and impact of processing on the bioactive compounds profile of eight non-melting peach cultivars, both in fresh and canned forms, thermally processed with either light syrup (LS) or grape juice (GJ) as the packing medium. LC–MS analysis demonstrated that the phytochemical profile is primarily genotype dependent. Prediction profile statistics revealed that cv. ‘Andross’, a predominant clingstone peach cultivar, possessed the highest desirability score among all the examined cultivars. Free zeaxanthin and lutein remained relatively unaffected by the canning process compared to free β-carotene, whereas soluble phenolic compounds, such as neochlorogenic acid, chlorogenic acid, procyanidin B1 and catechin, showed a large decrease following canning. Using GJ, an additional source of polyphenols, as packing medium led to a reduction of losses for the bioactive compounds. Overall, the experimental findings demonstrated that, besides the thermal degradation of some sensitive to heating compounds, there is a bidirectional flow of the bioactive compounds between the fruit tissue and the liquid matrix, as balanced out by diffusion processes

Hb A₂ Episkopi – a novel <i>δ</i>-globin chain variant [HBD:c.428C>T] in a family of mixed Cypriot–Lebanese descent

<p><b>Objectives:</b> Thalassaemia is a potentially lethal inherited anaemia, caused by reduced or absent synthesis of globin chains. Measurement of the minor adult haemoglobin Hb A₂, combining α- with δ-globin, is critical for the routine diagnosis of carrier status for α- or β-thalassaemia. Here, we aim to characterize a novel δ-globin variant, Hb A₂ Episkopi, in a single family of mixed Lebanese and Cypriot ancestry with mild hypochromic anaemia and otherwise normal globin genotype, which also presents with a coincidental 0.78-Mb sequence duplication on chromosome 1 (1q44) and developmental abnormalities.</p> <p><b>Methods</b>: Analyses included comprehensive haematological analyses, cation-exchange high-performance liquid chromatography (CE-HPLC), cellulose acetate electrophoresis (CAE), Sanger sequencing and structure-based stability predictions for Hb A₂ Episkopi.</p> <p><b>Results</b>: The GCT > GTT missense mutation, underlying Hb A₂ Episkopi, HBD:c.428C > T, introduces a cd142 codon change in the mature protein, resulting in reduced normal Hb A₂ amounts and a novel, less abundant Hb A₂ variant (HGVS: HBD:p.A143V), detectable as a delayed peak by CE-HPLC. The latter was in line with structure-based stability predictions, which indicated that the substitution of a marginal, non-helical and non-interface residue, five amino acids from the δ-globin chain carboxy-terminus, was moderately destabilizing.</p> <p><b>Discussion</b>: Detection of the new variant depends on the diagnostic set-up and had failed by CAE and on an independent CE-HPLC system, which, in unfavourable circumstances, may lead to misdiagnoses of β-thalassaemia as α-thalassaemia. Given the mixed background of the affected family, the ethnic origin of the mutation is unclear, and this study thus suggests awareness for possible detection of Hb A₂ Episkopi in both the Cypriot and the Lebanese populations.</p

Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders

Abstract Gene therapy using recombinant adeno‐associated virus (rAAV) vectors to treat blinding retinal dystrophies has become clinical reality. Therapeutically impactful targeting of photoreceptors still relies on subretinal vector delivery, which detaches the retina and harbours substantial risks of collateral damage, often without achieving widespread photoreceptor transduction. Herein, we report the development of novel engineered rAAV vectors that enable efficient targeting of photoreceptors via less invasive intravitreal administration. A unique in vivo selection procedure was performed, where an AAV2‐based peptide‐display library was intravenously administered in mice, followed by isolation of vector DNA from target cells after only 24 h. This stringent selection yielded novel vectors, termed AAV2.GL and AAV2.NN, which mediate widespread and high‐level retinal transduction after intravitreal injection in mice, dogs and non‐human primates. Importantly, both vectors efficiently transduce photoreceptors in human retinal explant cultures. As proof‐of‐concept, intravitreal Cnga3 delivery using AAV2.GL lead to cone‐specific expression of Cnga3 protein and rescued photopic cone responses in the Cnga3−/− mouse model of achromatopsia. These novel rAAV vectors expand the clinical applicability of gene therapy for blinding human retinal dystrophies

Variations in rates of severe perineal tears and episiotomies in 20 European countries: a study based on routine national data in Euro-Peristat Project

Introduction: Rates of severe perineal tears and episiotomies are indicators of obstetrical quality of care, but their use for international comparisons is complicated by difficulties with accurate ascertainment of tears and uncertainties regarding the optimal rate of episiotomies. We compared rates of severe perineal tears and episiotomies in European countries and analysed the association between these two indicators. Material and methods: We used aggregate data from national routine statistics available in the Euro-Peristat project. We compared rates of severe (third- and fourth-degree) tears and episiotomies in 2010 by mode of vaginal delivery (n = 20 countries), and investigated time trends between 2004 and 2010 (n = 9 countries). Statistical associations were assessed with Spearman's ranked correlations (rho). Results: In 2010 in all vaginal deliveries, rates of severe tears ranged from 0.1% in Romania to 4.9% in Iceland, and rates of episiotomies from 3.7% in Denmark to 75.0% in Cyprus. A negative correlation between the rates of episiotomies and severe tears was observed in all deliveries (rho = −0.66; p = 0.001), instrumental deliveries (rho = −0.67; p = 0.002) and non-instrumental deliveries (rho = −0.72; p < 0.001). However there was no relation between time trends of these two indicators (rho = 0.43; p = 0.28). Conclusions: The large variations in severe tears and episiotomies and the negative association between these indicators in 2010 show the importance of improving the assessment and reporting of tears in each country, and evaluating the impact of low episiotomy rates on the perineum.SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe