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Retinitis pigmentosa is a common cause of inherited blindness in adults, which in many cases is associated with an increase in the formation of reactive oxygen species (ROS) that induces DNA damage, triggering Poly-ADP-Ribose Polymerase 1 (PARP1) activation and leading to parthanatos-mediated cell death. Previous studies have shown that resveratrol (RSV) is a promising molecule that can mitigate PARP1 overactivity, but its low bioavailability is a limitation for medical use. This study examined the impact of a synthesized new acylated RSV prodrug, piceid octanoate (PIC-OCT), in the 661W cell line against H2O2 oxidative stress and in rd10 mice. PIC-OCT possesses a better ADME profile than RSV. In response to H2O2, 661W cells pretreated with PIC-OCT preserved cell viability in more than 38% of cells by significantly promoting SIRT1 nuclear translocation, preserving NAD+/NADH ratio, and suppressing intracellular ROS formation. These effects result from expressing antioxidant genes, maintaining mitochondrial function, reducing PARP1 nuclear expression, and preventing AIF nuclear translocation. In rd10 mice, PIC-OCT inhibited PAR-polymer formation, increased SIRT1 expression, significantly reduced TUNEL-positive cells in the retinal outer nuclear layer, preserved ERGs, and enhanced light chamber activity (all p values < 0.05). Our findings corroborate that PIC-OCT protects photoreceptors by modulating the SIRT1/PARP1 axis in models of retinal degeneration.

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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.

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BACKGROUND: Gene therapy is a therapeutic possibility for retinitis pigmentosa (RP), in which therapeutic transgenes are currently delivered to the retina by adeno-associated viral vectors (AAVs). Although their safety and efficacy have been demonstrated in both clinical and preclinical settings, AAVs present some technical handicaps, such as limited cargo capacity and possible immunogenicity in repetitive doses. The development of alternative, non-viral delivery platforms like nanoparticles is of great interest to extend the application of gene therapy for RP. METHODS: Amino-functionalized mesoporous silica-based nanoparticles (N-MSiNPs) were synthesized, physico-chemically characterized, and evaluated as gene delivery systems for human cells in vitro and for retinal cells in vivo. Transgene expression was evaluated by WB and immunofluorescence. The safety evaluation of mice subjected to subretinal injection was assessed by ophthalmological tests (electroretinogram, funduscopy, tomography, and optokinetic test). RESULTS: N-MSiNPs delivered transgenes to human cells in vitro and to retinal cells in vivo. No adverse effects were detected for the integrity of the retinal tissue or the visual function of treated eyes. N-MSiNPs were able to deliver a therapeutic transgene candidate for RP, PRPF31, both in vitro and in vivo. CONCLUSIONS: N-MSiNPs are safe for retinal delivery and thus a potential alternative to viral vectors.

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Printed circuit board (PCB) technology is well known, reliable, and low-cost, and its application to biomedicine, which implies the integration of microfluidics and electronics, has led to Lab-on-PCB. However, the biocompatibility of the involved materials has to be examined if they are in contact with biological elements. In this paper, the solder mask (PSR-2000 CD02G/CA-25 CD01, Taiyo Ink (Suzhou) Co., Ltd., Suzhou, China) of a commercial PCB has been studied for retinal cultures. For this purpose, retinal explants have been cultured over this substrate, both on open and closed systems, with successful results. Cell viability data shows that the solder mask has no cytotoxic effect on the culture allowing the application of PCB as the substrate of customized microelectrode arrays (MEAs). Finally, a comparative study of the biocompatibility of the 3D printer Uniz zSG amber resin has also been carried out.

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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.

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Mutations in the EYS gene are one of the major causes of autosomal recessive retinitis pigmentosa. EYS-retinopathy presents a severe clinical phenotype, and patients currently have no therapeutic options. The progress in personalised medicine and gene and cell therapies hold promise for treating this degenerative disease. However, lack of understanding and incomplete comprehension of disease's mechanism and the role of EYS in the healthy retina are critical limitations for the translation of current technical advances into real therapeutic possibilities. This review recapitulates the present knowledge about EYS-retinopathies, their clinical presentations and proposed genotype-phenotype correlations. Molecular details of the gene and the protein, mainly based on animal model data, are analysed. The proposed cellular localisation and roles of this large multi-domain protein are detailed. Future therapeutic approaches for EYS-retinopathies are discussed.

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Retinal dystrophies associated to mutations in the CRB1 gene comprise a wide array of clinical presentations. A blood sample from a patient with a family history of CRB1-retinal dystrophy was used to prepare the iPSC line ESi082-A. The genotype of the donor, affected of a perifoveal-bilateral macular dystrophy includes one frameshift deletion and one hypomorphic allele. ESi082-A cell line has been characterized for pluripotency and will be used to prepare retinal cellular models to study the dysfunction leading to the disease.

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Human induced pluripotent stem cells (hiPSCs) represent the most promising biological material for regenerative medicine applications. In this work, a 3D solid nanofibrous matrix of defined composition (Colamigel-S) consisting of 97 wt % gelatin, 2.6 wt % atelocollagen, and 0.4 wt % laminin has been reproducibly processed and characterized and exhibits a homogeneous nanofibrillar network of high surface area, interconnected microcavities, and typical D-periodic collagen fibril nanostructural features. The purpose of the study was to test the performance of Colamigel-S as substrate for in vitro hiPSCs culture, finding that these cells efficiently attach and grow keeping their characteristic stem morphology and undifferentiated state.

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It has been reported that resveratrol (RES) has a therapeutic effect in different neurodegenerative and ocular diseases. However, RES is rapidly eliminated from the organism, and high doses need to be administered resulting in potential toxic side effects. We hypothesized that a RES prodrug such as 3,4'-diglucosyl resveratrol (JC19) would reduce RES metabolism to produce a neuroprotective effect. Here, we have examined the protective effect of JC19 in an experimental mouse model of autosomal recessive RP. Rd10 mice at postnatal day 13 (P13) were subretinally injected with vehicle and two different doses of JC19. Electroretinogram (ERG) and histological evaluation were performed 15 days after injections. The amplitude of a- and b-waves was quantified in ERG recordings, and the number of photoreceptor nuclei in the outer nuclear layer was counted. In addition, the mouse retinas were immunostained with anti-rhodopsin antibodies. JC19 treatment delayed the loss of rod photoreceptor in rd10 mice, maintaining the expression of rhodopsin and preserving their electrical responses to light stimuli. The exact mechanism by which RES delays retinal degeneration in rd10 mice remains to be elucidated, but Sirtuin 1 activation could be one of the key molecular pathways involved in its neuroprotective effect.

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Age-related macular degeneration (AMD) is the leading cause of adult blindness in developed countries and is characterized by progressive degeneration of the macula, the central region of the retina. A human induced pluripotent stem cell (hiPSC) line was derived from peripheral blood mononuclear cells (PBMCs) from a patient with a clinical diagnosis of dry AMD carrying the CFH Y402H polymorphism. Sendai virus was using for reprogramming and the pluripotent and differentiation capacity of the cells were assessed by immunocytochemistry and RT-PCR.

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BACKGROUND: Testicular cancer (TCa) is a malignant tumor with highest incidence and mortality rates in Chile. The genes coding for cytochrome P450, glutathione-S-transferases (GSTs), and UDP glucuronyl transferases (UGT) participate in the biotransformation and detoxification of xenobiotics. Mutations in these genes have been associated with a high incidence of various types of cancer and an increased risk of presenting adverse reactions to drugs. OBJECTIVE: The aim of this study was to relate the presence of genetic polymorphisms in cytochrome P450 1A1 (CYP1A1), CYP3A4, GSTM1, GSTP1, GSTT1, and UGT1A1 genes and nongenetic factors with the risk of developing TCa. METHODS: A total of 276 volunteers from the Chilean general population and 251 Chilean TCa patients were recruited for the study. Genotypic analyses were performed using qPCR and PCR-RFLP. RESULTS: Variant alleles found to increase the risk of developing TCa were CYP1A1*2C (rs1048943), GSTP1 (rs1695), and GSTT1null, especially when in conjunction with a cancer family history and/or a smoking habit. The results of the multivariate analysis showed that the presence of variant alleles of GSTP1 (rs1695) together with a smoking habit and a family history of cancer accounted for a 15.9% risk of developing TCa in the Chilean population. CYP1A1*2C, GSTM1null, GSTT1null, and GSTP1 (rs1695) are statistically related to the risk of appearance of TCa, alone or associated with nongenetic factors. CONCLUSION: Therefore, phase I and II variant alleles might be useful in evaluating susceptibility to TCa in the studied population.

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IMPACT STATEMENT: In the promising field of cellular therapy for retinal degenerative diseases, a new biomaterial is proposed as a scaffold to grow and surgically introduce a monolayer of retinal pigment epithelial cells into the subretinal space, keeping the orientation of the cells for a proper functional integration of the transplant. The use of induced pluripotent stem cells as the starting material for retinal pigment epithelial cells is intended to advance toward a personalized medicine approach.

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Testicular cancer is one of the most commonly occurring malignant tumors in young men with fourfold higher rate of incidence and threefold higher mortality rates in Chile than the average global rates. Surgery is the initial line of treatment for testicular cancers, and is generally followed by chemotherapy, usually with combinations of bleomycin, etoposide, and cisplatin (BEP). However, the adverse effects of chemotherapy vary significantly among individuals; therefore, the present study explored the association of functionally significant allelic variations in genes related to the pharmacokinetics/pharmacodynamics of BEP and DNA repair enzymes with chemotherapy-induced toxicity in BEP-treated testicular cancer patients. We prospectively recruited 119 patients diagnosed with testicular cancer from 2010 to 2017. Genetic polymorphisms were analyzed using PCR and/or qPCR with TaqMan ®probes. Toxicity was evaluated based on the Common Terminology Criteria for Adverse Events, v4.03. After univariate analyses to define more relevant genetic variants (p < 0.2) and clinical conditions in relation to severe (III-IV) adverse drug reactions (ADRs), stepwise forward multivariate logistic regression analyses were performed. As expected, the main severe ADRs associated with the non-genetic variables were hematological (neutropenia and leukopenia). Univariate statistical analyses revealed that patients with ERCC2 rs13181 T/G and/or CYP3A4 rs2740574 A/G genotypes are more likely to develop alopecia; patients with ERCC2 rs238406 C/C genotype may develop leukopenia, and patients with GSTT1-null genotype could develop lymphocytopenia (III-IV). Patients with ERCC2 rs1799793 A/A were at risk of developing severe anemia. The BLMH rs1050565 G/G genotype was found to be associated with pain, and the GSTP1 G/G genotype was linked infection (p < 0.05). Multivariate analysis showed an association between specific ERCC1/2 genotypes and cumulative dose of BEP drugs with the appearance of severe leukopenia and/or febrile neutropenia. Grades III-IV vomiting, nausea, and alopecia could be partly explained by the presence of specific ERCC1/2, MDR1, GSTP1, and BLMH genotypes (p < 0.05). Hence, we provide evidence for the usefulness of pharmacogenetics as a tool for predicting severe ADRs in testicular cancer patients treated with BEP chemotherapy.

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PRPF31 gene codes for a ubiquitously expressed splicing factor but mutations affect exclusively the retina, producing the progressive death of photoreceptor cells. We have identified a novel PRPF31 mutation in a patient with autosomal dominant retinitis pigmentosa. A blood sample was obtained and mononuclear cells were reprogrammed using the non-integrative Sendai virus to generate the cell line CABi001-A. The iPSC line has been characterized for pluripotency and differentiation capacity and will be differentiated toward photoreceptors and retinal pigment epithelium cells to study the molecular mechanism of the disease and test possible therapeutic strategies.

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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.

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Cancer is the second leading cause of death in the world, causing 8.8 million deaths in 2015 according to the World Health Organization (WHO). Risk factors for cancer include smoking and alcohol con sumption. In Chile, 33.6% of the population and 21.2% of young people smokes. Alcohol consump tion in the Chilean population is 74.5% and 12.2% in young people. Among the physiological factors that influence the development of cancer, the genetic factor plays a relevant role. It has been shown that the presence of genetic polymorphisms that alter the ability of the body to eliminate contami nants increase the risk of developing cancer. The same applies to polymorphisms that prevent DNA repair due to damage caused by environmental pollutants such as cigarette smoke. The objective of this review is to analyze the state of the art of the relationship between pharmacogenetics, smoking, and alcohol consumption as risk factors for the development of cancer. In conclusion, the results suggest that the presence of polymorphisms that alter the function of biotransformation enzymes phase I (CYP1A1, CYP1E1) and phase II (GST), as well as polymorphisms in DNA repair enzymes (ERCC1 / ERCC2), increase the risk of cancer induced by smoking and alcohol consumption. This association is important considering that smoking and drinking alcohol are highly prevalent among the Chilean population.

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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.

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El cáncer es la segunda causa de muerte en el mundo, según datos de la Organización Mundial de la Salud (OMS) en el año 2015 ocasionó 8,8 millones de muertes. Dentro de los factores de riesgo para el desarrollo de cáncer se encuentran el tabaquismo y el consumo de alcohol. En Chile el 33,6% de la población fuma y un 21,2 % de los jóvenes. El consumo de alcohol en la población chilena es de 74,5 % y en los jóvenes de un 12,2 %. Entre los factores fisiológicos que influyen en el desarrollo de cáncer, el factor genético juega un rol relevante, habiéndose demostrado que la presencia de polimorfismos genéticos alteran la capacidad del organismo de eliminar contaminantes y aumentan el riesgo de desarrollar cáncer. Lo mismo ocurre con polimorfismos que impiden la reparación de ADN debido a daños producidos por efecto de contaminantes ambientales como el humo de cigarrillo. El objetivo de esta revisión es analizar el estado del arte de la relación entre farmacogenética, tabaco y alcohol como factores de riesgo para el desarrollo de cáncer. Los resultados sugieren que la presencia de po limorfismos que alteran la función de enzimas de biotransformación fase I (CYP1A1, CYP1E1) y fase II (GST), además de polimorfismos en enzimas de reparación del ADN (ERCC1/ERCC2) aumentan el riesgo de cáncer inducido por el hábito tabáquico y alcohólico. Esta asociación es importante, si consideramos que en la población chilena el hábito de fumar y beber alcohol es altamente prevalente.

Cancer is the second leading cause of death in the world, causing 8.8 million deaths in 2015 according to the World Health Organization (WHO). Risk factors for cancer include smoking and alcohol con sumption. In Chile, 33.6% of the population and 21.2% of young people smokes. Alcohol consump tion in the Chilean population is 74.5% and 12.2% in young people. Among the physiological factors that influence the development of cancer, the genetic factor plays a relevant role. It has been shown that the presence of genetic polymorphisms that alter the ability of the body to eliminate contami nants increase the risk of developing cancer. The same applies to polymorphisms that prevent DNA repair due to damage caused by environmental pollutants such as cigarette smoke. The objective of this review is to analyze the state of the art of the relationship between pharmacogenetics, smoking, and alcohol consumption as risk factors for the development of cancer. In conclusion, the results suggest that the presence of polymorphisms that alter the function of biotransformation enzymes phase I (CYP1A1, CYP1E1) and phase II (GST), as well as polymorphisms in DNA repair enzymes (ERCC1 / ERCC2), increase the risk of cancer induced by smoking and alcohol consumption. This association is important considering that smoking and drinking alcohol are highly prevalent among the Chilean population.

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Retinitis pigmentosa (RP) is the most common cause of inherited blindness in adults. Mutations in the PRPF31 gene produce autosomal dominant RP (adRP). To date there are no effective treatments for this disease. The purpose of this study was to design an efficient non-viral vector for human PRPF31 gene delivery as an approach to treat this form of adRP. Span based nanoparticles were developed to mediate gene transfer in the subretinal space of a mouse model of adRP carrying a point mutation (A216P) in the Prpf31 gene. Funduscopic examination, electroretinogram, optomotor test and optical coherence tomography were conducted to further in vivo evaluate the safety and efficacy of the nanosystems developed. Span-polyarginine (SP-PA) nanoparticles were able to efficiently transfect the GFP and PRPF31 plasmid in mice retinas. Statistically significant improvement in visual acuity and retinal thickness were found in Prpf31A216P/+ mice treated with the SP-PA-PRPF31 nanomedicine.

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