'Ex vivo' gene correction of PRPF31 c.165G>A mutation causing retinitis pigmentosa

Motivation: Retinitis pigmentosa (RP) is the most common form of retinal dystrophy, a group of blinding diseases characterized by progressive photoreceptor death, with a prevalence of 1 in 4000. RP is highly-heterogeneous, with 15% of autosomal dominant cases caused by mutations in the pre-mRNA processing factors (PRPFs), components of the spliceosome.To date, there are no effective treatments for RP. Gene editing is a rapidly evolving field that may in the future, allow the repair of a mutated endogenous locus. CRISPR/Cas9 system has a mechanism of action based on nucleotide recognition of target DNA by engineered single-guide RNA (sgRNA) and Cas9 endonuclease activity. Genomic edition of patient-derived induced pluripotent stem cells (iPSCs) would allow autologous transplantation of repaired cells, once differentiated to retinal cell types.Methods: iPSCs obtained from a RP patient with a PRPF31 c.165G>A mutation were the starting biological material. Pluripotency of the iPSCs was checked by inmunofluorescence (IF) analysis.Disease phenotyping of the cell line was performed by IF for PRPF31 and for the ciliary protein ARL13B, as PRPF31 mutations have been previously described to affect cilia.sgRNAs directed to the mutation were designed using the web crispor.tefor.net. The best sgRNA and a ssODN template, covering the mutation site, were synthesized by IDT. The sgRNA-CRISPR/Cas9 complex was assembled and co-transfected with the ssODN into the iPSCs. FACs was used to measure the efficiency and to select transfected cells. A bulk transfected cell population was analyzed by Sanger sequencing to check for HR-mediated knock-in. Selection of individual iPSC clones and genotyping is being performed to search  for corrected clones.Results: Positive labeling for OCT4, NANOG, SSEA3, SSEA4 and TRA-1-81 showed pluripotency of the iPSC line. PRPF31 immunolocalization and quantificacion have been used to phenotype the iPSC line compared to a healthy control. Sanger sequencing of the genomic DNA showed successful editing of the mutation in the bulk population of transfected cells. Different culture conditions were tested for iPSC clonal selection. Best conditions provided a 0.8 % of efficiency. The CRISPR/Cas9-corrected iPSC clone will be differentiated to retinal pigment epithelium (RPE) and photoreceptors, in parallel with uncorrected PRPF31-iPSCs, to establish if in situ gene editing restores key celular and functional phenotypes associated with this type of RP

Characterization of retinal cells derived from iPSCs of a patient with PRPF31 associated retinitis pigmentosa

Motivation: Retinitis pigmentosa (RP) is a group of hereditary retinal dystrophies caused by mutations in different genes with a prevalence of 1 in 4000. It is an untreatable disease with a variable clinical evolution in which patients develop severe visual impairment or total blindness. Mutations in pre-mRNA splicing PRPF31 gene have been described as the second most common cause of autosomal dominant RP. Previous studies relate mutations in PRPF31 with dysfunction and degeneration of the retinal pigment epithelium (RPE). Thanks to the ability to obtain and differentiate induced pluripotent stem cells (iPSC), retinal models can be generated to study the disease mechanism and to evaluate new therapies. This work is based on a personalized cellular model obtained by differentiating RPE from iPSCs of a patient with PRPF31 c.165G mutation, which will be used to study the mechanism of the disease. Methods: iPSCs and previously differentiated RPE cells have been cultured and imaged. The characterization of the RNA level expression of specific genes of both cell types has been performed by RT-PCR. Expression at the protein level has been analyzed by Western blot. At the physiological level, the ability of the cellular model to establish an epithelial barrier has been evaluated by transepithelial electrical resistance (TER). Results: Phase contrast images showed a characteristic and distintive morphology of iPSC and RPE cells. RT-PCR showed the silencing of pluripotency genes such as NANOG in RPE cells, as well as the exclusive expression of specific genes such as CRALBP and RPE65 in RPE. In the comparative study of the cellular models of patient and healthy control, it was observed a variation in the expression levels of the PRPF31 and RPE65 genes. In Western blot, the PRPF31 protein detected in the patient's RPE showed a different band pattern compared with healthy control and iPSCs. Finally, TER showed a similar maturation of the two cell models compared, indicating that PRPF31 c.165G mutation does not affect the cells adhesions. Conclusions: The cellular model of RPE with PRPF31 c.165G mutation has been correctly differentiated, allowing the study of the consequences at the cellular level of this genetic defect. The decrease found in RPE65 gene expression suggests that this could be the mechanism by wich PRPF31 c.165G mutation causes RP, because RPE65 insufficiency is a known cause of blindness

Generation of a human iPS cell line from a patient with retinitis pigmentosa due to EYS mutation

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease. Mutations in EYS have been associated with autosomal recessive RP. The human iPS cell line, CABi002-A, derived from peripheral blood mononuclear cells from a patient carrying a heterozygous double mutation in EYS gene was generated by non-integrative reprogramming technology, using hOCT3/4, hSOX2, hc-MYC and hKLF4 reprogramming factors. Pluripotency and differentiation capacity were assessed by immunocytochemistry and RT-PCR. This iPSC line can be further differentiated towards the affected cells to understand the pathophysiology of the disease and test new therapeutic strategies.Cellex FoundationFundación Progreso y Salu

Site-directed Mutagenesis of Cytochromec 6 from Synechocystissp. PCC 6803

This paper reports the first site-directed mutagenesis analysis of any cytochrome c 6, a heme protein that performs the same function as the copper-protein plastocyanin in the electron transport chain of photosynthetic organisms. Photosystem I reduction by the mutants of cytochromec 6 from the cyanobacteriumSynechocystis sp. PCC 6803 has been studied by laser flash absorption spectroscopy. Their kinetic efficiency and thermodynamic properties have been compared with those of plastocyanin mutants from the same organism. Such a comparative study reveals that aspartates at positions 70 and 72 in cytochrome c 6 are located in an acidic patch that may be isofunctional with the well known “south-east” patch of plastocyanin. Calculations of surface electrostatic potential distribution in the mutants of cytochromec 6 and plastocyanin indicate that the changes in protein reactivity depend on the surface electrostatic potential pattern rather than on the net charge modification induced by mutagenesis. Phe-64, which is close to the heme group and may be the counterpart of Tyr-83 in plastocyanin, does not appear to be involved in the electron transfer to photosystem I. In contrast, Arg-67, which is at the edge of the cytochrome c 6 acidic area, seems to be crucial for the interaction with the reaction center.Dirección General de Investigación Científica y Técnica (DGICYT, Grant PB96-1381)European Union (EU, CHRX-CT94-0540 and ERB-FMRX-CT98-0218)Junta de Andalucía (PAI, CVI-0198

Obtención y caracterización de células de pluripotencia inducida a partir de monocitos humanos

La obtención de células de pluripotencia inducida (iPS) a partir de células somáticas ha proporcionado al campo de la medicina regenerativa una nueva herramienta para la terapia celular, evitando los problemas éticos relacionados con las células madre embrionarias (ESC) (Ronen et al. 2012). Las iPS, al igual que las ESC, son capaces de diferenciarse hacia distintos tipos celulares por lo que pueden derivarse a modelos celulares de enfermedades, apropiados tanto para la investigación básica como para el ensayo de nuevas terapias. La fuente habitual de iPS son fibroblastos obtenidos de una biopsia de piel. En nuestro caso, el objetivo es poner a punto un sistema de generación de modelos celulares a partir de sangre periférica, que es el tejido adulto más accesible, por lo que la obtención de muestras es mucho menos invasivo, y además permite el acceso a numerosas muestras almacenadas en los bancos de sangre (Phillips et al. 2012). Concretamente, en nuestro proyecto pretendemos obtener epitelio pigmentario de la retina (RPE) a partir de monocitos de pacientes con degeneración macular (AMD) portadores de un genotipo de susceptibilidad a la enfermedad. La metodología seguida ha consistido en el cultivo de monocitos sanguíneos y su transformación a iPS mediante la expresión de factores de reprogramación introducidos en la célula mediante el virus Sendai (SeV). El SeV es un virus no integrativo y se emplea como portador de los factores de transcripción claves para activar la pluripotencia, es decir, permite la expresión de transgenes sin el riesgo de modificación del genoma del hospedador (Fusaki et al. 2009). Además, la eficiencia de generación de iPS con el SeV es significativamente mayor que con otros métodos como la reprogramación por mARN o mediante vectores episomales.Los clones de iPS obtenidos, antes de su diferenciación hacia RPE, han de ser caracterizados mediante la comprobación de la ausencia del SeV (por PCR), la expresión de marcadores de pluripotencia, mediante inmunocitoquímica y PCR (OCT4, SOX2, SSEA-4, NANOG) y ensayos de fosfatasa alcalina. Los resultados obtenidos nos permiten comprobar que la reprogramación ha sido satisfactoria.Hasta el momento, hemos sido capaces de reprogramar células sanguíneas hacia iPS y mantenerlas. El siguiente paso será empezar a optimizar los protocolos de diferenciación hacia RPE. Tras caracterizarlo procederemos a realizar todo el proceso de reprogramación y diferenciación con muestras de pacientes con AMD

Retinal pigment epithelium degeneration caused by aggregation of PRPF31 and the role of HSP70 family of proteins

Background Mutations in pre-mRNA splicing factor PRPF31 can lead to retinitis pigmentosa (RP). Although the exact disease mechanism remains unknown, it has been hypothesized that haploinsufficiency might be involved in the pathophysiology of the disease. Methods In this study, we have analyzed a mouse model containing the p.A216P mutation in Prpf31 gene. Results We found that mutant Prpf31 protein produces cytoplasmic aggregates in the retinal pigment epithelium and decreasing the protein levels of this splicing factor in the nucleus. Additionally, normal protein was recruited in insoluble aggregates when the mutant protein was overexpressed in vitro. In response to protein aggregation, Hspa4l is overexpressed. This member of the HSP70 family of chaperones might contribute to the correct folding and solubilization of the mutant protein, allowing its translocation to the nucleus. Conclusions Our data suggests that a mechanism haploinsufficiency and dominant-negative is involved in retinal degeneration due to mutations in PRPF31. HSP70 over-expression might be a new therapeutic target for the treatment of retinal degeneration due to PRPF31 mutations.This project has been financed through a) The ISCIII (Miguel Servet-I, 2015), co-financed by the European Regional Development Fund (ERDF), No CP15/00071. b) The European Union’s Horizon 2020 research and innovation program, under grant agreement No 634479. c) Regional Ministry of Economy, Innovation and Science of the Junta de Andalucía, No P09-CTS-04967.info:eu-repo/semantics/publishedVersio

Caracterización de PRPF31

Motivación: La retinitis pigmentosa es un grupo heterogéneo de distrofias retinales caracterizada por una progresiva degeneración de los fotorreceptores, que acaba produciendo deficiencia visual o ceguera. PRPF31 es uno de los genes cuya mutación causa este tipo de distrofia. Aunque la proteína Prpf31 es un factor de splicing presente en todo el organismo, su mutación sólo produce distrofia retinal. Actualmente no se conoce el mecanismo implicado en la enfermadad, por lo que intentaremos discernirlo con ayuda de este proyecto.Métodos: Utilizando la secuencia del gen PRPF31, planteamos clonarla en el vector p3XFLAG y transfectar con la construcción células de mamífero. La fusión con FLAG facilitará los estudios de localización subcelular y de las interacciones por inmunoprecipitación. Transfectaremos con la construcción las líneas celulare RPE1, derivada de epitelio pigmentario de la retina (RPE) humana y 293, para estudiar la localización y las interacciones de Prpf31 con otras proteínas, con el objetivo de estudiar la base molecular de la enfermedad y poder desarrollar terapias génicas, farmacológicas y/o celulares. Resultados: La clonación de la secuencia de PRPF31 aún está en proceso ya que no hemos obtenido la construcción diseñada a pesar de probar diversos métodos. Se han empezado a poner a punto las condiciones de transfección usando otra construcción similar, tanto con la línea 293 como con RPE1. Se ha estudiado la distribución de Prpf31 en retina y RPE de ratón por western blot, mostrando una mayor abundancia en RPE, lo que no es común a otros factores de splicing como el PRPF3 y PRPF8, con los que se ha comparado. Conclusiones: PRPF31 debe tener alguna función específica en RPE, aún por definir, por lo que la degeneración de los fotorreceptores será secundaria a una disfunción del RPE. Hay otros casos en los que un gen presenta una función específicamente diferente en la retina, como en el caso de ATR, que causa síndrome de Seckel. ATR es un controlador de la división celular, pero en la retina este gen es crítico para el desarrollo postnatal de los fotorreceptores, por lo que su mutación causa degeneraciónen la retina

Analysis of gene network bifurcation during optic cup morphogenesis in zebrafish

Sight depends on the tight cooperation between photoreceptors and pigmented cells, which derive from common progenitors through the bifurcation of a single gene regulatory network into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identified upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebrafish. We analyze cis-regulatory modules and enriched transcription factor motives to show extensive network redundancy and context-dependent activity. We identify downstream targets, highlighting an early recruitment of desmosomal genes in the flattening RPE and revealing Tead factors as upstream regulators. We investigate the RPE specification network dynamics to uncover an unexpected sequence of transcription factors recruitment, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counseling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases.This work is supported by the following grants: (I) To J.-R.M.-M.: From the Spanish Ministry of Science, Innovation, and Universities (MICINN): BFU2017-86339P with FEDER funds, MDM-2016-0687 and PY20_00006/Junta de Andalucía. (II) To O.B. Australian Research Council (ARC) Discovery Project (DP190103852). (III) To F.-J.D.-C.: Andalusian Ministry of Health, Equality and Social Policies (PI-0099-2018). (IV) To P.B.: BFU2016-75412-R with FEDER funds; PCIN-2015-176-C02-01/ERA-Net Neuron ImprovVision, and a CBMSO Institutional grant from the Fundación Ramón Areces. (V) To both J.-R.M.-M. and P.B.: BFU2016-81887-REDT, as well as Fundación Ramón Areces-2016 (Supporting L.B.)

Cost-effective sequence analysis of 113 genes in 1,192 probands with retinitis pigmentosa and Leber congenital amaurosis

Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.This study received funding from Novartis. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. This work was supported by grants from Foundation Fighting Blindness Career Development Award CDGE-0621-0809-RAD (SR), Foundation Fighting Blindness project program award PPA-0123-0841-UCL (SR and SdB), Retinitis Pigmentosa Fighting Blindness, Fight for Sight UK (RP Genome Project GR586), Ghent University Special Research Fund (BOF20/GOA/023) (EDB and BL); EJP RD Solve-RET EJPRD19-234 (EDB, BL, SB, CR, FC, and SR). EDB (1802220N) and BL (1803816N) are FWO Senior Clinical Investigators of the Research Foundation Flanders (FWO). EDB, BL, SB, FC, and SR are members of ERN-EYE (Framework Partnership Agreement No. 739534)

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente