Inherited retinal dystrophies (IRDs) affect an estimated 1 in every 2000 people, this corresponding to nearly 2 million cases worldwide. Currently, 270 genes have been associated with IRDs, most of them altering the function of photoreceptors and retinal pigment epithelium. Gene therapy has been proposed as a potential tool for improving visual function in these patients. Clinical trials in animal models and humans have been successful in various types of IRDs. Recently, voretigene neparvovec (Luxturna®) has been approved by the US Food and Drug Administration for the treatment of biallelic mutations in the RPE65 gene. The current state of the art in gene therapy involves the delivery of various types of viral vectors into the subretinal space to effectively transduce diseased photoreceptors and retinal pigment epithelium. For this, subretinal injection is becoming increasingly popular among researchers and clinicians. To date, several approaches for subretinal injection have been described in the scientific literature, all of them effective in accessing the subretinal space. The growth and development of gene therapy give rise to the need for a standardized procedure for subretinal injection that ensures the efficacy and safety of this new approach to drug delivery. The goal of this review is to offer an insight into the current subretinal injection techniques and understand the key factors in the success of this procedure.

Inherited retinal dystrophies (IRDs) are a group of rare retinal conditions, including retinitis pigmentosa (RP), caused by monogenic mutations in 1 out of more than 250 genes. Despite recent advancements in gene therapy, there is still a lack of an effective treatment for this group of retinal conditions. MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNAs that inhibit gene expression. Control of miRNAs-mediated protein expression has been described as a widely used mechanism for post-transcriptional regulation in many physiological and pathological processes in different organs, including the retina. Our main purpose was to test the hypothesis that modulation of a group of miRNAs can protect photoreceptor cells from death in the rd10 mouse model of retinitis pigmentosa. For this, we incorporated modulators of three miRNAs in adeno-associated viruses (AAVs), which were administered through sub-retinal injections. The results obtained indicate that inhibition of the miR-6937-5p slows down the visual deterioration of rd10 mice, reflected by an increased electroretinogram (ERG) wave response under scotopic conditions and significant preservation of the outer nuclear layer thickness. This work contributes to broadening our knowledge on the molecular mechanisms underlying retinitis pigmentosa and supports the development of novel therapeutic approaches for RP based on miRNA modulation.

Hepatocellular carcinoma (HCC) pathogenesis is associated with alterations in splicing machinery components (spliceosome and splicing factors) and aberrant expression of oncogenic splice variants. We aimed to analyze the expression and potential role of the spliceosome component PRPF8 (pre-mRNA processing factor 8) in HCC. PRPF8 expression (mRNA/protein) was analyzed in a retrospective cohort of HCC patients (n = 172 HCC and nontumor tissues) and validated in two in silico cohorts (TCGA and CPTAC). PRPF8 expression was silenced in liver cancer cell lines and in xenograft tumors to understand the functional and mechanistic consequences. In silico RNAseq and CLIPseq data were also analyzed. Our results indicate that PRPF8 is overexpressed in HCC and associated with increased tumor aggressiveness (patient survival, etc.), expression of HCC-related splice variants, and modulation of critical genes implicated in cancer-related pathways. PRPF8 silencing ameliorated aggressiveness in vitro and decreased tumor growth in vivo. Analysis of in silico CLIPseq data in HepG2 cells demonstrated that PRPF8 binds preferentially to exons of protein-coding genes, and RNAseq analysis showed that PRPF8 silencing alters splicing events in multiple genes. Integrated and in vitro analyses revealed that PRPF8 silencing modulates fibronectin (FN1) splicing, promoting the exclusion of exon 40.2, which is paramount for binding to integrins. Consistent with this finding, PRPF8 silencing reduced FAK/AKT phosphorylation and blunted stress fiber formation. Indeed, HepG2 and Hep3B cells exhibited a lower invasive capacity in membranes treated with conditioned medium from PRPF8-silenced cells compared to medium from scramble-treated cells. This study demonstrates that PRPF8 is overexpressed and associated with aggressiveness in HCC and plays important roles in hepatocarcinogenesis by altering FN1 splicing, FAK/AKT activation and stress fiber formation.

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The intrinsic heterogeneity of endocrine-related cancers (ERCs) hampers the identification and development of global and effective therapeutic treatments for these pathologies. However, the dysregulation of the splicing process has been postulated as a new common hallmark shared by most cancer types, since it is associated with the appearance of splicing variants with oncogenic potential (e.g. CD44v6, BCL-xs, AR-v7, SST5TMD4, In1-ghrelin). Yet, the putative alteration, pathophysiological role and potential therapeutic utility of the elements involved in the control of the splicing process [i.e. spliceosome components (SCs) and splicing factors (SFs)] remain still unknown. For this reason, we have analysed the expression levels of a representative set of SCs (n=18) and SFs (n=27) in different cohorts of ERCs [i.e. growth hormone secreting pituitary tumors (n=96); non-functioning pituitary tumors (n=23); pancreatic neuroendocrine tumors (n=20); prostate cancer (n=126); and in four in silico cohorts of liver cancer (HCC; n=445, n=115, n=75, n=45)]. Our results showed that the SF3b subunit 1 ( SF3B1 ) gene, which encodes a protein necessary for spliceosome assembly, was consistently overexpressed in all the ERCs evaluated in this study. Interestingly, SF3B1 expression was positively correlated and associated with clinical and molecular aggressiveness features (e.g. histopathological grade, presence of metastasis, expression of oncogenic splicing variants, etc.) in these ERCs. In vitro analyses revealed that the specific blockade of SF3B1 activity, using the pladienolide-B compound, exhibited potent and relevant antitumor effects (reducing cell proliferation, migration, tumorospheres and colonies formation, hormone release and inducing apoptosis) in the vast majority of representative models of ERC cells tested herein (primary ERCs cell cultures and cell lines such as BON-1, QGP-1, LNCaP, 22Rv1, PC-3, HepG2, Hep3b or SNU-387). Remarkably, the antitumor effects of pladienolide-B treatment were further validated in vivo in that we found a significant reduction of tumor volume after 9-days of local treatment of pladienolide-B in a xenograft model of ERC. Moreover, we found that pladienolide-B treatment modulated an ample repertoire of molecular events, such as inhibition of major oncogenic signalling pathways (e.g. PI3K/AKT and JNK), modulation of the expression of key tumour markers (e.g. MKI67 / CDK6 / CDKN2A ) and oncogenic splicing variants (e.g. AR-v7/In1-ghrelin ), and regulation of the expression pattern of key components of mRNA homeostasis-associated machineries (spliceosome and SURF/EJC). In conclusion, these results indicate that SF3B1 is consistently overexpressed in different ERCs and associated to malignant features and that its pharmacological blockade with pladienolide-B could...