Characterization of the Positive Transcription Regulator PfaR for Improving Eicosapentaenoic Acid Production in Shewanella putrefaciens W3-18-1.

The biosynthetic pathway of eicosapentaenoic acid (EPA) has previously been reported in marine bacteria, while the regulatory mechanism remains poorly understood. In this study, a putative transcriptional regulator PfaR encoded adjacent to the PFA biosynthesis gene cluster (pfaEABCD) was computationally and experimentally characterized. Comparative analyses on the wild type (WT) strain, in-frame deletion, and overexpression mutants revealed that PfaR positively regulated EPA synthesis at low temperature. RNA-Seq and real-time quantitative PCR analyses demonstrated that PfaR stimulated the transcription of pfaABCD. The transcription start site of pfaR was mapped by using primer extension and highly conserved promoter motifs bound by the housekeeping Sigma 70 factor that were identified in the upstream of pfaR. Moreover, overexpression of PfaR in WT strain W3-18-1 at low temperature could improve EPA productivity from 0.07% to 0.13% (percentage of EPA to dry weight, mg/mg) of dry weight. Taken together, these findings could provide important implications into the transcriptional control and metabolic engineering in terms of EPA productivity for industrial strains. IMPORTANCE We have experimentally confirmed that PfaR is a positive transcription regulator that promotes EPA synthesis at low temperature in Shewanella putrefaciens W3-18-1. Overexpression of PfaR in WT strain W3-18-1 could lead to a 1.8-fold increase in EPA productivity at low temperature. It is further shown that PfaR may be regulated by housekeeping Sigma 70 factor at low temperature.

Ribosomal RNA Modulates Aggregation of the Podospora Prion Protein HET-s.

The role of the nucleic acids in prion aggregation/disaggregation is becoming more and more evident. Here, using HET-s prion from fungi Podospora anserina (P. anserina) as a model system, we studied the role of RNA, particularly of different domains of the ribosomal RNA (rRNA), in its aggregation process. Our results using Rayleigh light scattering, Thioflavin T (ThT) binding, transmission electron microscopy (TEM) and cross-seeding assay show that rRNA, in particular the domain V of the major rRNA from the large subunit of the ribosome, substantially prevents insoluble amyloid and amorphous aggregation of the HET-s prion in a concentration-dependent manner. Instead, it facilitates the formation of the soluble oligomeric "seeds", which are capable of promoting de novo HET-s aggregation. The sites of interactions of the HET-s prion protein on domain V rRNA were identified by primer extension analysis followed by UV-crosslinking, which overlap with the sites previously identified for the protein-folding activity of the ribosome (PFAR). This study clarifies a missing link between the rRNA-based PFAR and the mode of propagation of the fungal prions.

Efficient episomal gene transfer to human hepatic cells using the pFAR4-S/MAR vector.

Liver-directed gene therapy, using mainly viral vectors for the genetic cell modification, is a promising therapeutic approach for many genetic and metabolic liver diseases. The recent successful preclinical trials with AAV vectors expose the benefits as well as the limitations of the system. We focused on the development of an alternative non-viral episomal gene transfer system, by inserting the DNA element Scaffold/Matrix Attachment Region (S/MAR) into the free of antibiotic resistance gene miniplasmid vector (pFAR4). We produced pFAR4 derivative experimental vectors, carrying the eGFP gene driven by the composite HCRHPi liver-specific promoter and either lacking (pFAR4-noS/MAR) or containing the S/MAR element in an upstream (pFAR-S/MAR-IN) or downstream (pFAR4-S/MAR-OUT) configuration in relation to the poly-A signal of the eGFP expression cassette. Upon transfer into Huh7 cells by lipofection, vector pFAR4-S/MAR IN showed significantly higher transfection efficiency and eGFP expression than the control vector or the pFAR4-S/MAR-OUT (p < 0.005), estimated by fluorescent microscopy and flow cytometry. Stable transfections were produced only with cultures containing vector pFAR4-S/MAR IN, through the expansion of single colonies, which displayed sustained GFP expression and plasmid copy number per cell of 2.3 ± 0.4, at 3 months of culture. No vector integration events were detected in these cultures by FISH analysis, while the presence of free, circular plasmids was documented by plasmid rescue assay. The presence of S/MAR renders pFAR4 miniplasmid substantially more efficient regarding episomal gene transfer and is suitable for liver-directed studies towards gene therapy applications.

lncRNA PFAR Promotes Lung Fibroblast Activation and Fibrosis by Targeting miR-138 to Regulate the YAP1-Twist Axis.

Long non-coding RNAs (lncRNAs) have been reported to be involved in various pathophysiological processes in many diseases. However, the role and mechanism of lncRNAs in idiopathic pulmonary fibrosis (IPF) have not been explicitly delineated. In the present study, we reported that lncRNA NONMMUT065582, designated pulmonary fibrosis-associated RNA (PFAR), is upregulated in the lungs of mice with lung fibrosis as well as in fibrotic lung fibroblasts. Overexpression of PFAR promoted fibrogenesis through modulation of miR-138, whereas knockdown of PFAR attenuated TGF-ß1-induced fibrogenesis in lung fibroblasts. In addition, knockdown of miR-138 promoted fibrogenesis by targeting regulation of yes-associated protein 1 (YAP1), whereas enhanced expression of miR-138 attenuated fibrogenesis in lung fibroblasts. Mechanistically, PFAR acted as competing endogenous RNA (ceRNA) of miR-138: forced expression of PFAR reduced the expression and activity of miR-138 to activate YAP1 and promote fibrogenesis in lung fibroblasts, whereas loss of YAP1 abrogated the pro-fibrotic effect of PFAR. More importantly, PFAR silencing alleviated BLM-induced lung fibrosis in mice. Taken together, the results of our study identified lncRNA PFAR as a new pro-fibrotic molecule that acts as a ceRNA of miR-138 during lung fibrosis and demonstrated PFAR as a novel therapeutic target for the prevention and treatment of lung fibrosis.

LncRNA PFAR facilitates the proliferation and migration of papillary thyroid carcinoma by competitively binding to miR-15a.

Papillary thyroid carcinoma (PTC) is type of aggressive tumor, with a markedly declined survival rate when distant metastasis occurs. It is of great significance to develop potential biomarkers to evaluate the progression of PTC. LncRNAs are recently widely claimed with biomarker value in malignant tumors. Herein, the role of LncRNA PFAR in PTC was investigated to explore potential prognostic marker for PTC. Compared to NTHY-ORI 3-1 cells, LncRNA PFAR was found markedly upregulated in PTC cell lines. In LncRNA PFAR knockdown TPC-1 cells, markedly declined cell viability, increased apoptotic rate, enhancive number of migrated cells, and elevated migration distance were observed, accompanied by a suppressed activity of the RET/AKT/mTOR signaling. In LncRNA PFAR overexpressed BCPAP cells, signally increased cell viability, declined apoptotic rate, reduced number of migrated cells, decreased migration distance, and increased tumor volume and tumor weight in nude mice xenograft model were observed, accompanied by an activation of the RET/AKT/mTOR signaling. The binding site between LncRNA PFAR and miR-15a, as well as miR-15a and RET, was confirmed by the dual luciferase reporter assay. The FISH study revealed that LncRNA PFAR was mainly located in the cytoplasm. Furthermore, the impact of the siRNA targeting LncRNA PFAR against the growth and migration of PTC cells was abolished by the inhibitor of miR-15a or SC79, an activator of AKT/mTOR signaling. Collectively, LncRNA PFAR facilitated the proliferation and migration of PTC cells by mediating the miR-15a/RET axis.

Anti-prion Drugs Targeting the Protein Folding Activity of the Ribosome Reduce PABPN1 Aggregation.

Prion diseases are caused by the propagation of PrPSc, the pathological conformation of the PrPC prion protein. The molecular mechanisms underlying PrPSc propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrPSc propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide and metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrPSc propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies.

Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver.

Non-viral gene delivery into the liver generally mediates a transient transgene expression. A comparative analysis was performed using two gene vectors, pFAR4 and pKAR4, which differ by the absence or presence of an antibiotic resistance marker, respectively. Both plasmids carried the same eukaryotic expression cassette composed of a sulfamidase (Sgsh) cDNA expressed from the human alpha antitrypsin liver-specific promoter. Hydrodynamic injection of the pFAR4 construct resulted in prolonged sulfamidase secretion from the liver, whereas delivery of the pKAR4 construct led to a sharp decrease in circulating enzyme. After induction of hepatocyte division, a rapid decline of sulfamidase expression occurred, indicating that the pFAR4 derivative was mostly episomal. Quantification analyses revealed that both plasmids were present at similar copy numbers, whereas Sgsh transcript levels remained high only in mice infused with the pFAR4 construct. Using a chromatin immunoprecipitation assay, it was established that the 5' end of the expression cassette carried by pKAR4 exhibited a 7.9-fold higher heterochromatin-to-euchromatin ratio than the pFAR4 construct, whereas a bisulfite treatment did not highlight any obvious differences in the methylation status of the two plasmids. Thus, by preventing transgene expression silencing, the pFAR4 gene vector allows a sustained transgene product secretion from the liver.

Microbubbles for Nucleic Acid Delivery in Liver Using Mild Sonoporation.

Ultrasound-mediated gene delivery is an interesting approach, which could help in increasing gene transfer in deep tissues. Moreover, it allows for performing experiments guided by the image to determine which elements are required. Microbubbles complexed with a eukaryotic expression cassette are excellent agents as they are responsive to ultrasounds and, upon oscillation, can destabilize membranes to enhance gene transfer. Here, we describe the preparation of positively charged microbubbles, plasmid free of antibiotic resistance marker, their combination and the conditions of ultrasound-mediated liver transfection post-systemic administration in mice. This association allowed us to obtain a superior liver gene expression at least over 8 months after a single injection.

Improved molecular platform for the gene therapy of rare diseases by liver protein secretion.

Many rare monogenic diseases are treated by protein replacement therapy, in which the missing protein is repetitively administered to the patient. However, in several cases, the missing protein is required at a high and sustained level, which renders protein therapy far from being adequate. As an alternative, a gene therapy treatment ensuring a sustained effectiveness would be particularly valuable. Liver is an optimal organ for the secretion and systemic distribution of a therapeutic transgene product. Cutting edge non-viral gene therapy tools were tested in order to produce a high and sustained level of therapeutic protein secretion by the liver using the hydrodynamic delivery technique. The use of S/MAR matrix attachment region provided a slight, however not statistically significant, increase in the expression of a reporter gene in the liver. We have selected the von Willebrand Factor (vWF) gene as a particularly challenging large gene (8.4 kb) for liver delivery and expression, and also because a high vWF blood concentration is required for disease correction. By using the optimized miniplasmid pFAR free of antibiotic resistance gene together with the Sleeping Beauty transposon and the hyperactive SB100X transposase, we have obtained a sustainable level of vWFblood secretion by the liver, at 65% of physiological level. Our results point to the general use of this plasmid platform using the liver as a protein factory to treat numerous rare disorders by gene therapy.

The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells.

The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells.

The double life of the ribosome: When its protein folding activity supports prion propagation.

It is no longer necessary to demonstrate that ribosome is the central machinery of protein synthesis. But it is less known that it is also key player of the protein folding process through another conserved function: the protein folding activity of the ribosome (PFAR). This ribozyme activity, discovered more than 2 decades ago, depends upon the domain V of the large rRNA within the large subunit of the ribosome. Surprisingly, we discovered that anti-prion compounds are also potent PFAR inhibitors, highlighting an unexpected link between PFAR and prion propagation. In this review, we discuss the ancestral origin of PFAR in the light of the ancient RNA world hypothesis. We also consider how this ribosomal activity fits into the landscape of cellular protein chaperones involved in the appearance and propagation of prions and other amyloids in mammals. Finally, we examine how drugs targeting the protein folding activity of the ribosome could be active against mammalian prion and other protein aggregation-based diseases, making PFAR a promising therapeutic target for various human protein misfolding diseases.

Long-Term PEDF Release in Rat Iris and Retinal Epithelial Cells after Sleeping Beauty Transposon-Mediated Gene Delivery.

Pigment epithelium derived factor (PEDF) is a potent antiangiogenic, neurotrophic, and neuroprotective molecule that is the endogenous inhibitor of vascular endothelial growth factor (VEGF) in the retina. An ex vivo gene therapy approach based on transgenic overexpression of PEDF in the eye is assumed to rebalance the angiogenic-antiangiogenic milieu of the retina, resulting in growth regression of choroidal blood vessels, the hallmark of neovascular age-related macular degeneration. Here, we show that rat pigment epithelial cells can be efficiently transfected with the PEDF-expressing non-viral hyperactive Sleeping Beauty transposon system delivered in a form free of antibiotic resistance marker miniplasmids. The engineered retinal and iris pigment epithelium cells secrete high (141 ± 13 and 222 ± 14 ng) PEDF levels in 72 hr in vitro. In vivo studies showed cell survival and insert expression during at least 4 months. Transplantation of the engineered cells to the subretinal space of a rat model of choroidal neovascularization reduces almost 50% of the development of new vessels.

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids.

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal blood vessels growing into the subretinal space, leading to retinal pigment epithelial (RPE) cell degeneration and vision loss. Vessel growth results from an imbalance of pro-angiogenic (e.g., vascular endothelial growth factor [VEGF]) and anti-angiogenic factors (e.g., pigment epithelium-derived factor [PEDF]). Current treatment using intravitreal injections of anti-VEGF antibodies improves vision in about 30% of patients but may be accompanied by side effects and non-compliance. To avoid the difficulties posed by frequent intravitreal injections, we have proposed the transplantation of pigment epithelial cells modified to overexpress human PEDF. Stable transgene integration and expression is ensured by the hyperactive Sleeping Beauty transposon system delivered by pFAR4 miniplasmids, which have a backbone free of antibiotic resistance markers. We demonstrated efficient expression of the PEDF gene and an optimized PEDF cDNA sequence in as few as 5 × 103 primary cells. At 3 weeks post-transfection, PEDF secretion was significantly elevated and long-term follow-up indicated a more stable secretion by cells transfected with the optimized PEDF transgene. Analysis of transgene insertion sites in human RPE cells showed an almost random genomic distribution. The results represent an important contribution toward a clinical trial aiming at a non-viral gene therapy of nvAMD.

Spectroscopic and DFT studies on 6-aminophenanthridine and its derivatives provide insights in their activity towards ribosomal RNA.

6-Aminophenanthridine (6AP), a plant alkaloid possessing antiprion activity, inhibits ribosomal RNA dependent protein folding activity of the ribosome (referred as PFAR). We have compared 6AP and its three derivatives 6AP8Cl, 6AP8CF3 and 6APi for their activity in inhibition of PFAR. Since PFAR inhibition requires 6AP and its derivatives to bind to the ribosomal RNA (rRNA), we have measured the binding affinity of these molecules to domain V of 23S rRNA using fluorescence spectroscopy. Our results show that similar to the antiprion activity, both the inhibition of PFAR and the affinity towards rRNA follow the order 6AP8CF3 > 6AP8Cl > 6AP, while 6APi is totally inactive. To have a molecular insight for the difference in activity despite similarities in structure, we have calculated the nucleus independent chemical shift using first principles density functional theory. The result suggests that the deviation of planarity in 6APi and steric hindrance from its bulky side chain are the probable reasons which prevent it from interacting with rRNA. Finally, we suggest a probable mode of action of 6AP, 6AP8CF3 and 6AP8Cl towards rRNA.