Search

Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases1. Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation2–7. However, tRNA-based gene therapy has not yielded an optimal combination of clinical efficacy and safety and there is presently no treatment for individuals with nonsense mutations. Here we introduce a strategy based on altering native tRNAs into efficient suppressor tRNAs (sup-tRNAs) by individually fine-tuning their sequence to the physico-chemical properties of the amino acid that they carry. Intravenous and intratracheal lipid nanoparticle (LNP) administration of sup-tRNA in mice restored the production of functional proteins with nonsense mutations. LNP–sup-tRNA formulations caused no discernible readthrough at endogenous native stop codons, as determined by ribosome profiling. At clinically important PTCs in the cystic fibrosis transmembrane conductance regulator gene (CFTR), the sup-tRNAs re-established expression and function in cell systems and patient-derived nasal epithelia and restored airway volume homeostasis. These results provide a framework for the development of tRNA-based therapies with a high molecular safety profile and high efficacy in targeted PTC suppression.

Spine surgery outcomes assessment currently relies on patient-reported outcome measures, which satisfy established reliability and validity criteria, but are limited by the inherently subjective and discrete nature of data collection. Physical activity measured from smartphones offers a new data source to assess postoperative functional outcomes in a more objective and continuous manner.To present a methodology to characterize preoperative mobility and gauge the impact of surgical intervention using objective activity data garnered from smartphone-based accelerometers.Smartphone mobility data from 14 patients who underwent elective lumbar decompressive surgery were obtained. A time series analysis was conducted on the number of steps per day across a 2-year perioperative period. Five distinct clinical stages were identified using a data-driven approach and were validated with clinical documentation.Preoperative presentation was correctly classified as either a chronic or acute mobility decline in 92% of patients, with a mean onset of acute decline of 11.8 ± 2.9 weeks before surgery. Postoperative recovery duration demonstrated wide variability, ranging from 5.6 to 29.4 weeks (mean: 20.6 ± 4.9 weeks). Seventy-nine percentage of patients ultimately achieved a full recovery, associated with an 80% ± 33% improvement in daily steps compared with each patient's preoperative baseline (P = .002). Two patients subsequently experienced a secondary decline in mobility, which was consistent with clinical history.The perioperative clinical course of patients undergoing spine surgery was systematically classified using smartphone-based mobility data. Our findings highlight the potential utility of such data in a novel quantitative and longitudinal surgical outcome measure.

PDF file - 114K, Supplemental Figure 5 Cellular distribution of HIF-2alpha under normal oxygen conditions. (a) Representative confocal microscopy images (magnification 400x) showing HIF-2α or Nox4 immunofluorescense, DAPI nuclear staining, or merged images. b) Control (NS) or Nox4 shRNA (KD) cells were imaged following exposure to TEMPOL (0.25 mM), DTT (1mM). c) 786-0 NS cells imaged following transduction with Ad-GFP or Ad-MnSOD. d) 786-0 cells expressing pcDNA-Nox4 imaged with or without treatment with TEMPOL (0.25 mM).

PDF file - 376K, Supplemental Figure 2. Characterization of 786-0 cells stably transfected with the human, full-length Nox4 cDNA. (a) Morphology of 786-0 cells following exogenous expression of Nox4 reveals smaller, denser, more-rounded appearance relative to parental 786-0 cells. (b) Cell viability assay. Equal numbers of untransfected cells or 786-0 Nox4 cells were plated in 96-well plates. After attachment, assay reagent was added, incubated at 37{degree sign}C, and the fluorescent signal read at 590 nm.

PDF file - 224K, Supplemental Figure 1. Quantitative RT-PCR (a) and semi-quantitative RT-PCR (b) for detection of Nox1 in 786-0, RCC4 or LNCaP prostate cancer cells using two published primer pairs(14, 17),. Error bar represents +/- SE.

PDF file - 183K, Supplemental Figure 4. Cytotoxicity was measured by CellTiter-Blue (Promega, Madison, WI). 786-0 NS cells were plated at 1 x 104 cells per well in 96-well plates. After 24 hours, dithiothreitol was added as indicated in 5 replicate wells. 20 �l of the CellTiter-Blue reagent was added to each well and viability quantitated by spectrofluorometer (Spectra Max Plus384, Molecular Devices) and expressed as percentage of viability of media-only controls. Error bars indicate +/- S.E. mean.

Most sporadically occurring renal tumors include a functional loss of the tumor suppressor von Hippel Lindau (VHL). Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia-inducible factor-2α (HIF2α), a master transcriptional regulator of genes that drive diverse processes, including angiogenesis, proliferation, and anaerobic metabolism. In determining the critical functions for HIF2α expression in RCC cells, the NADPH oxidase NOX4 has been identified, but the pathogenic contributions of NOX4 to RCC have not been evaluated directly. Here, we report that NOX4 silencing in VHL-deficient RCC cells abrogates cell branching, invasion, colony formation, and growth in a murine xenograft model RCC. These alterations were phenocopied by treatment of the superoxide scavenger, TEMPOL, or by overexpression of manganese superoxide dismutase or catalase. Notably, NOX4 silencing or superoxide scavenging was sufficient to block nuclear accumulation of HIF2α in RCC cells. Our results offer direct evidence that NOX4 is critical for renal tumorigenesis and they show how NOX4 suppression and VHL re-expression in VHL-deficient RCC cells are genetically synonymous, supporting development of therapeutic regimens aimed at NOX4 blockade. Cancer Res; 74(13); 3501–11. ©2014 AACR.

PDF file - 98K, Supplemental Figure 3. Lucigenin chemiluminescence assay for superoxide detection performed as described in materials and methods on a) isolated membrane fractions from parental 786-0, RCC4 and Caki-1 cells or isolated cell fractions from 786-0 (b) or RCC4 (c) cells. RLU: relative light units.

PDF file - 42K

PDF file - 446K, Supplemental Figure 6 Immunohistochemistry for Nox4 and HIF-2alpha in 786-0 NS and KD xenograft explants confirms re-expression of Nox4 in KD tumors with corresponding elevated HIF-2alpha expression. Magnification 200X.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as Lumacaftor (VX-809), Tezacaftor (VX-661) and Elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry (AP-MS) multiplexed with isobaric Tandem Mass Tags (TMT) was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knock-downs sensitize P67L to VX-445 and further enhance the correction of this variant. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize unresponsive CFTR variants to known and available correctors.

Purine nucleoside phosphorylase (PNP) gene transfer represents a promising approach to treatment of head and neck malignancies. We tested recombinant adenovirus already in phase I/II clinical testing and leading-edge patient-derived xenografts (PDX) as a means to optimize this therapeutic strategy.Our experiments investigated purine base cytotoxicity, PNP enzyme activity following treatment of malignant tissue, tumor mass regression, viral receptor studies, and transduction by tropism-modified adenovirus.Replication deficient vector efficiently transduced PDX cells and mediated significant anticancer effect following treatment with fludarabine phosphate in vivo. Either 6-methylpurine or 2-fluoroadenine (toxic molecules generated by the PNP approach) ablated head and neck cancer cell proliferation. High levels of adenovirus-3 specific receptors were detected in human tumor models, and vector was evaluated that utilizes this pathway.Our studies provide the scientific foundation necessary to improve PNP prodrug cleavage and advance a new treatment for head and neck cancer.

The aim is to develop and evaluate rizatriptan benzoate (RB) loaded nanostructured lipid carriers (NLCs) that might improve oral bioavailability. NLC was prepared using High-Speed Homogenization (HSH) coupled with the ultrasonication method. Process parameters were selected on basis of the trial-and-error method which is time for homogenization, speed of homogenization, and time for sonication. Using a Design expert, 32 Full Factorial design was prepared using solid lipid to liquid lipid ratio and concentration of surfactant as independent variables. RB-NLC was evaluated on basis of particle size, %Entrapment Efficiency (EE), Zeta potential, Transmission electron microscopy (TEM), In-Vitro permeation study, and Ex-vivo permeation study. Confocal Laser Scanning Microscopy was performed to study the uptake of RB-NLC through M-Cells, and Peyer's Patches. RB-NLC dispersion was converted into free-flowing lyophilized powder using cryoprotectant which is trehalose and in vitro permeation study was performed. Among all the batches of RB-NLC, the Optimized batch was evaluated. The optimized formulation showed a particle size of 412.8 ± 1.15nm with 0.261 PDI and − 36.6mV zeta potential. It showed 84.26 ± 1.03% EE. TEM image showed a spherical shape and smooth surface area. In-Vitro and Ex-vivo studies showed 97.21 ± 0.36%CDR up to 22 hours & 61.02 ± 1.25%CDR up to 6 hours. Lyophilized powder showed 96.37 ± 0.36%CDR up to 22 hours. Based on the data we can conclude that RB-NLC increases the permeation and it may increase its Bioavailability (BA). Lyophilized powder of RB-NLC showed a sustained release pattern and it may reduce the dosing frequency of the drug.