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

'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

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

Citocromo c6 y plastocianina de la cianobacteria Synechocystis PCC 6803: estudio de la relación estructura-función en las proteínas nativas y modificadas por mutagénesis dirigida

El estudio de los mecanismos de reconocimiento molecular y transferencia de electrones entre proteína se ha abordado en este trabajo usando como modelo una parte de la cadena fotosintética de transporte de electrones de la cianobacteria Synechocystis sp