Circular RNA (circRNA) molecules have critical functions during brain development and in brain-related disorders. Here, we identified and validated a circRNA, circHTT(2,3,4,5,6), stemming from the Huntington's disease (HD) gene locus that is most abundant in the central nervous system (CNS). We uncovered its evolutionary conservation in diverse mammalian species, and a correlation between circHTT(2,3,4,5,6) levels and the length of the CAG-repeat tract in exon-1 of HTT in human and mouse HD model systems. The mouse orthologue, circHtt(2,3,4,5,6), is expressed during embryogenesis, increases during nervous system development, and is aberrantly upregulated in the presence of the expanded CAG tract. While an IRES-like motif was predicted in circHTT(2,3,4,5,6), the circRNA does not appear to be translated in adult mouse brain tissue. Nonetheless, a modest, but consistent fraction of circHtt(2,3,4,5,6) associates with the 40S ribosomal subunit, suggesting a possible role in the regulation of protein translation. Finally, circHtt(2,3,4,5,6) overexpression experiments in HD-relevant STHdh striatal cells revealed its ability to modulate CAG expansion-driven cellular defects in cell-to-substrate adhesion, thus uncovering an unconventional modifier of HD pathology.
- Publikační typ
- časopisecké články MeSH
PURPOSE: To assess the safety and feasibility of direct vitrectomy-sparing subretinal injection for gene delivery in a large animal model. METHODS: The experimental Liběchov minipigs were used for subretinal delivery of a plasmid DNA vector (pS/MAR-CMV-copGFP) with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP) reporter (copGFP) and a scaffold/matrix attachment region (S/MAR) sequence. The eyes were randomized to subretinal injection of the vector following pars plana vitrectomy (control group) or a direct injection without prior vitrectomy surgery (experimental group). Intra- and post-operative observations up to 30 days after surgery were compared. RESULTS: Six eyes of three mini-pigs underwent surgery for delivery into the subretinal space. Two eyes in the control group were operated with a classical approach (lens-sparing vitrectomy and posterior hyaloid detachment). The other four eyes in the experimental group were injected directly with a subretinal cannula without vitrectomy surgery. No adverse events, such as endophthalmitis, retinal detachment and intraocular pressure elevation were observed post-operatively. The eyes in the experimental group had both shorter surgical time and recovery while achieving the same surgical goal. CONCLUSIONS: This pilot study demonstrates that successful subretinal delivery of gene therapy vectors is achievable using a direct injection without prior vitrectomy surgery.
- MeSH
- genetická terapie * metody MeSH
- genetické vektory * aplikace a dávkování MeSH
- injekce nitrooční MeSH
- miniaturní prasata * MeSH
- modely nemocí na zvířatech MeSH
- pilotní projekty MeSH
- plazmidy aplikace a dávkování MeSH
- prasata MeSH
- retina MeSH
- studie proveditelnosti * MeSH
- technika přenosu genů * MeSH
- vitrektomie * metody MeSH
- zelené fluorescenční proteiny genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Age-related macular degeneration (AMD) is the most frequent cause of blindness in developed countries. The replacement of dysfunctional human retinal pigment epithelium (hRPE) cells by the transplantation of in vitro-cultivated hRPE cells to the affected area emerges as a feasible strategy for regenerative therapy. Synthetic biomimetic membranes arise as powerful hRPE cell carriers, but as biodegradability is a requirement, it also poses a challenge due to its limited durability. hRPE cells exhibit several characteristics that putatively respond to the type of membrane carrier, and they can be used as biomarkers to evaluate and further optimize such membranes. Here, we analyze the pigmentation, transepithelial resistance, genome integrity, and maturation markers of hRPE cells plated on commercial polycarbonate (PC) versus in-house electrospun polylactide-based (PLA) membranes, both enabling separate apical/basolateral compartments. Our results show that PLA is superior to PC-based membranes for the cultivation of hRPEs, and the BEST1/RPE65 maturation markers emerge as the best biomarkers for addressing the quality of hRPE cultivated in vitro. The stability of the cultures was observed to be affected by PLA aging, which is an effect that could be partially palliated by the coating of the PLA membranes.
- Publikační typ
- časopisecké články MeSH
We applied Raman spectroscopy to brain and skin tissues from a minipig model of Huntington's disease. Differences were observed between measured spectra of tissues with and without Huntington's disease, for both brain tissue and skin tissue. There are linked to changes in the chemical composition between tissue types. Using machine learning we correctly classified 96% of test spectra as diseased or wild type, indicating that the test would have a similar accuracy when used as a diagnostic tool for the disease. This suggests the technique has great potential in the rapid and accurate diagnosis of Huntington's and other neurodegenerative diseases in a clinical setting.
- MeSH
- Huntingtonova nemoc * diagnóza MeSH
- lidé MeSH
- miniaturní prasata MeSH
- mozek MeSH
- neurodegenerativní nemoci * MeSH
- prasata MeSH
- Ramanova spektroskopie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. METHODS: We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 μm pore size and prepared Poly (L-lactide-co-DL-lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 μm. RESULTS: Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real-time qPCR (RT-qPCR) and electron microscopy. RT-qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehyde-binding protein 1), RPE65 (retinal pigment epithelium-specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia-associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na+ /K+ ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of α-SMA proved the stability of the RPE polarization state and the absence of epithelial-to-mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. CONCLUSIONS: Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long-term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA-cultured RPEs are a plausible source for the replacement of non-functioning RPEs during cell therapy.
PURPOSE: The development of primary human retinal pigmented epithelium (hRPE) for clinical transplantation purposes on biodegradable scaffolds is indispensable. We hereby report the results of the subretinal implantation of hRPE cells on nanofibrous membranes in minipigs. METHODS: The hRPEs were collected from human cadaver donor eyes and cultivated on ultrathin nanofibrous carriers prepared via the electrospinning of poly(L-lactide-co-DL-lactide) (PDLLA). "Libechov" minipigs (12-36 months old) were used in the study, supported by preoperative tacrolimus immunosuppressive therapy. The subretinal implantation of the hRPE-nanofibrous carrier was conducted using general anesthesia via a custom-made injector during standard three-port 23-gauge vitrectomy, followed by silicone oil endotamponade. The observational period lasted 1, 2, 6 and 8 weeks, and included in vivo optical coherence tomography (OCT) of the retina, as well as post mortem immunohistochemistry using the following antibodies: HNAA and STEM121 (human cell markers); Bestrophin and CRALBP (hRPE cell markers); peanut agglutining (PNA) (cone photoreceptor marker); PKCα (rod bipolar marker); Vimentin, GFAP (macroglial markers); and Iba1 (microglial marker). RESULTS: The hRPEs assumed cobblestone morphology, persistent pigmentation and measurable trans-epithelial electrical resistance on the nanofibrous PDLLA carrier. The surgical delivery of the implants in the subretinal space of the immunosuppressed minipigs was successfully achieved and monitored by fundus imaging and OCT. The implanted hRPEs were positive for HNAA and STEM121 and were located between the minipig's neuroretina and RPE layers at week 2 post-implantation, which was gradually attenuated until week 8. The neuroretina over the implants showed rosette or hypertrophic reaction at week 6. The implanted cells expressed the typical RPE marker bestrophin throughout the whole observation period, and a gradual diminishing of the CRALBP expression in the area of implantation at week 8 post-implantation was observed. The transplanted hRPEs appeared not to form a confluent layer and were less capable of keeping the inner and outer retinal segments intact. The cone photoreceptors adjacent to the implant scaffold were unchanged initially, but underwent a gradual change in structure after hRPE implantation; the retina above and below the implant appeared relatively healthy. The glial reaction of the transplanted and host retina showed Vimentin and GFAP positivity from week 1 onward. Microglial activation appeared in the retinal area of the transplant early after the surgery, which seemed to move into the transplant area over time. CONCLUSIONS: The differentiated hRPEs can serve as an alternative cell source for RPE replacement in animal studies. These cells can be cultivated on nanofibrous PDLLA and implanted subretinally into minipigs using standard 23-gauge vitrectomy and implantation injector. The hRPE-laden scaffolds demonstrated relatively good incorporation into the host retina over an eight-week observation period, with some indication of a gliotic scar formation, and a likely neuroinflammatory response in the transplanted area despite the use of immunosuppression.
- Publikační typ
- časopisecké články MeSH
Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas-mediated gene repair or gene therapy in vitro. AAV-based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.
The review intends to overview a wide range of nanostructured natural, synthetic and biological membrane implants for tissue engineering to help in retinal degenerative diseases. Herein, we discuss the transplantation strategies and the new development of material in combination with cells such as induced pluripotent stem cells (iPSC), mature retinal cells, adult stem cells, retinal progenitors, fetal retinal cells, or retinal pigment epithelial (RPE) sheets, etc. to be delivered into the subretinal space. Retinitis pigmentosa and age-related macular degeneration (AMD) are the most common retinal diseases resulting in vision impairment or blindness by permanent loss in photoreceptor cells. Currently, there are no therapies that can repair permanent vision loss, and the available treatments can only delay the advancement of retinal degeneration. The delivery of cell-based nanostructure scaffolds has been presented to enrich cell survival and direct cell differentiation in a range of retinal degenerative models. In this review, we sum up the research findings on different types of nanostructure scaffolds/substrate or material-based implants, with or without cells, used to deliver into the subretinal space for retinal diseases. Though, clinical and pre-clinical trials are still needed for these transplants to be used as a clinical treatment method for retinal degeneration.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Huntingtin (HTT)-lowering therapies hold promise to slow down neurodegeneration in Huntington's disease (HD). Here, we assessed the translatability and long-term durability of recombinant adeno-associated viral vector serotype 5 expressing a microRNA targeting human HTT (rAAV5-miHTT) administered by magnetic resonance imaging-guided convention-enhanced delivery in transgenic HD minipigs. rAAV5-miHTT (1.2 × 1013 vector genome (VG) copies per brain) was successfully administered into the striatum (bilaterally in caudate and putamen), using age-matched untreated animals as controls. Widespread brain biodistribution of vector DNA was observed, with the highest concentration in target (striatal) regions, thalamus, and cortical regions. Vector DNA presence and transgene expression were similar at 6 and 12 months after administration. Expression of miHTT strongly correlated with vector DNA, with a corresponding reduction of mutant HTT (mHTT) protein of more than 75% in injected areas, and 30 to 50% lowering in distal regions. Translational pharmacokinetic and pharmacodynamic measures in cerebrospinal fluid (CSF) were largely in line with the effects observed in the brain. CSF miHTT expression was detected up to 12 months, with CSF mHTT protein lowering of 25 to 30% at 6 and 12 months after dosing. This study demonstrates widespread biodistribution, strong and durable efficiency of rAAV5-miHTT in disease-relevant regions in a large brain, and the potential of using CSF analysis to determine vector expression and efficacy in the clinic.
- MeSH
- genetická terapie MeSH
- genetické vektory genetika MeSH
- Huntingtonova nemoc * genetika terapie MeSH
- lidé MeSH
- mikro RNA * metabolismus MeSH
- miniaturní prasata metabolismus MeSH
- modely nemocí na zvířatech MeSH
- prasata MeSH
- protein huntingtin genetika metabolismus MeSH
- tkáňová distribuce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Genetically modified rodent models of Huntington's disease (HD) have been especially valuable to our understanding of HD pathology and the mechanisms by which the mutant HTT gene alters physiology. However, due to inherent differences in genetics, neuroanatomy, neurocircuitry and neurophysiology, animal models do not always faithfully or fully recapitulate human disease features or adequately predict a clinical response to treatment. Therefore, conducting translational studies of candidate HD therapeutics only in a single species (i.e. mouse disease models) may not be sufficient. Large animal models of HD have been shown to be valuable to the HD research community and the expectation is that the need for translational studies that span rodent and large animal models will grow. Here, we review the large animal models of HD that have been created to date, with specific commentary on differences between the models, the strengths and disadvantages of each, and how we can advance useful models to study disease pathophysiology, biomarker development and evaluation of promising therapeutics.
