tbuckley/cabot-search huggingface dataset:
576. DNA Ends Matter: The Impact of Using CRISPR/Cas9 Variants on DNA Repair Pathway Choices and Editing Profiles at the HBB Locus
Authors: Tanushree Phadke, Anne Bothmer, Christina Lee, Hayat Abdulkerim, Luis Barrera, S. Moss, Hari Jayaram, Cecilia Cotta-Ramusino
Journal: Molecular Therapy (2016)

Sickle Cell Anemia is an inherited recessive disorder caused by a single point mutation in the human beta globin (HBB) gene resulting in an abnormal type of hemoglobin. Here we report targeting the HBB locus using CRISPR/Cas9 technology for correction of Sickle Cell Disease, which affects nearly 1 million people worldwide. Cas9 and its variants can be used to introduce a variety of breaks including blunt double stranded break (DSB), single nicks, or dual nicks leaving either a 3' or 5' overhang. The type of cut and donor used can play a role in triggering different repair pathways, thus, resulting in various editing profiles. Using a single strand oligonucleotide (ssODN), we characterize different DNA repair outcomes including indel mutations resulting from Non Homologous End-Joining (NHEJ), Homology-Dependent Repair (HDR) using the donor as a template, and, finally, Gene Conversion (a kind of HDR event) using the closely related HBD gene as an endogenous template. Repair using homologous sequences from the HBD gene results in partial gene-conversion yielding a chimeric HBB-HBD gene that corrects the sickle cell point mutation. We observed that the Cas9 nickase/gRNA pair leaving a 5' overhang displayed a significantly higher frequency of gene conversion and gene correction than other Cas9-induced DNA end structures. We also provide evidence that overexpression or down-regulation of critical factors in the repair pathways influence the repair pathway balance. In summary, we demonstrate that the frequency of various repair outcomes under different conditions offers insight into the mechanisms of repair of Cas9-induced DNA cleavage. The data support a therapeutic approach in which correction of the sickle-cell mutation is efficiently mediated through HDR using a donor template or by gene-conversion using the endogenous HBD gene.
Compared Antitumor Activity of GA101 and Rituximab against the Human RL Follicular Lymphoma Xenografts in SCID Beige Mice.
Authors: Stéphane Dalle, Lina Reslan, Stéphanie Brunet Manquat, Franck Herting, Christian Klein, Pablo Umana, Charles Dumontet
Journal: Blood (2008)
Abstract LB-146: TGF-β-induced tumor heterogeneity and drug resistance of cancer stem cells
Authors: Naoki Oshimori, Daniel Oristian, Elaine Fuchs
Journal: Cancer Research (2015)

Abstract Among the most common and life-threatening cancers world-wide, squamous cell carcinoma (SCC) exhibit high rates of tumor recurrence following anti-cancer therapy. Subsets of cancer stem cells (CSCs) often escape anti-cancer therapeutics and promote recurrence. However, its sources and mechanisms that generate tumor heterogeneity and therapy-resistant cell population are largely unknown. Tumor microenvironment may drive intratumor heterogeneity by transmitting signaling factors, oxygen and metabolites to tumor cells depending on their proximity to the local sources. While the hypothesis is attractive, experimental evidence is lacking, and non-genetic mechanisms that drive functional heterogeneity remain largely unknown. As a potential non-genetic factor, we focused on TGF-β because of its multiple roles in tumor progression. Here we devise a functional reporter system to monitor, track and modify TGF-β signaling in mouse skin SCC in vivo. Using this approach, we found that perivascular TGF-β in the tumor microenvironment generates heterogeneity in TGF-β signaling in neighboring CSCs. This heterogeneity is functionally important: small subsets of TGF-β-responding CSCs proliferate more slowly than their non-responding counterparts. They also exhibit invasive morphology and a malignant differentiation program compared to their non-responding neighbors. By lineage tracing, we show that although TGF-β-responding CSCs clonally expand more slowly they gain a growth advantage in a remarkable ability to escape cisplatin-induced apoptosis. We show that indeed it is their progenies that make a substantial contribution in tumor recurrence. Surprisingly, the slower proliferating state of this subset of CSCs within the cancer correlated with but did not confer the survival advantage to anti-cancer drugs. Using transcriptomic, biochemical and genetic analyses, we unravel a novel mechanism by which heterogeneity in the tumor microenvironment allows a subset of CSCs to respond to TGF-β, and evade anti-cancer drugs. Our findings also show that TGF-β established ability to suppress proliferation and promote invasion and metastasis do not happen sequentially, but rather simultaneously. This new work build upon the roles of this factor in tumor progression, and sets an important paradigm for a non-genetic factor that produces tumor heterogeneity. Citation Format: Naoki Oshimori, Daniel Oristian, Elaine Fuchs. TGF-β-induced tumor heterogeneity and drug resistance of cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-146. doi:10.1158/1538-7445.AM2015-LB-146
Pediatric Burkitt’s lymphoma (diagnosis and treatment evaluation) in NCI, Pediatric Oncology Unit: Misurata/Libya initial experience.
Authors: E. I. Aldosouky, M. A. Elfagieh, A. O. Jebriel
Journal: Journal of Clinical Oncology (2011)

e20000 Background: In this study the patients are Burkitt’s lymphoma (BL) stage III, and treated by protocol consisting of alternating cycles: A and B, for 8 cycles. This protocol was adopted in NCI, Egypt. It has the advantage of short duration of therapy, no high dose methotrexate (MTX), no maintenance therapy or cranial irradiation and it has a good overall survival. Methods: This is a retrospective study included all patients (three) with pediatric BL collected over 2 years at the NCI Misurata/Pediat Oncol Unit from Nov 2008 to Nov 2010. Chemotherapy protocol: Cycle A (cyclophosphamide, adriamycin, vincristine, cytosar, plus intrathecal MTX and cytosar). Cycle B (ifosfamide, vepeside, MTX, vincristine and intrathecal cytosar and MTX. Intrathecals were given during the first 4 cycles. 1st case; 3 years-old girl presented with acute abdomen, ileocaecal intusussception and caecal mass. Surgical release of intussusception then right haemicolectomy was performed. 2nd case; 3 years old girl presented with buccal mass, kidney lesions, multiple abdomino-pelvic lymph nodes, and true cut biopsy from buccal mass and fine needle aspiration cytology from abdominal LNs were done. 3rd case; 18 years old boy presented with Thyroid nodule right lobe, bilateral cervical LNs, right renal nodule, Pancreatic tail nodule, and celio-mesentric LNs. He underwent partial thyroidectomy (right lobe).The three patients received 8 cycles of chemotherapy (A&B) with evaluation after 2 cycles and after 8 cycles. Results: All cases were Stage III, LDH < 500, None had bone marrow involvement or CNS infiltration. Complete evaluation was done after 8 cycles, revealed complete remission for the three patients. The median follow up duration was 11 months (range 6-22 months). Conclusions: Initial experience and review highlighted some unusual presentations of this tumor that should be kept in mind with differential diagnosis for similar presentations. Ileocecal with Burkitt’s lymphoma may mimic clinically acute appendicitis. Pathologic and immunohistochemical testing remain the only definitive methods of diagnosis. High dose methotrexate is not a must to improve outcome of non advanced Burkitt’s lymphoma.
495. In Vivo Evidence of trans-Splicing in a Humanized Mouse Model of Autosomal Dominant Retinitis Pigmentosa Induced By Mutation of the Rhodopsin Gene
Authors: Adeline Berger, Stéphanie Lorain, Charlène Joséphine, Mélissa Desrosiers, Cécile Peccate, Thomas Voit, Luis Garcia, José-Alain Sahel, Alexis-Pierre Bemelmans
Journal: Molecular Therapy (2015)

The most frequent cause of retinitis pigmentosa (RP), a group of hereditary retinal dystrophies leading to blindness, is the occurrence of point mutations in the Rhodopsin (RHO) gene. Most of these mutations lead to gain of functions or dominant negative effects deleterious for photoreceptors, thus resulting in a dominant mode of transmission. Moreover, it has been shown that variations in RHO expression level are also deleterious for the retina. Gene transfer strategies modifying RHO mutations should therefore lead both to suppression of mutant protein expression and restoration of the normal one at a physiological level. Spliceosome-mediated RNA trans-splicing should in theory respect these constraints by repairing mutations at the pre-mRNA level. Using this technology, the expression level of the repaired mRNA will indeed depend solely on that of the gene endogenous promoter. To achieve this goal, this approach consists of introducing, by gene transfer, an exogenous RNA – called PTM, for Pre-Trans-splicing Molecule – able to bind the pre-mRNA and promote splicing in trans, leading to replacement of the mutant part of the RHO pre-mRNA. We engineered fourteen different RHO-PTMs able to repair any mutation in RHO exons 2, 3, 4 and 5, which differ only on their binding sequence to RHO pre-mRNA. To determine the efficiency of each PTM, we transiently co-transfected HEK293T cells with a plasmid encoding a PTM and another encoding the wild-type or mutant RHO. The maximum trans-splicing efficiency observed at the mRNA level was about 25%. We improved this efficiency to 40% by refinement of the PTM sequence, through reintroduction of an endogenous RHO intron in the cDNA part of the PTM. We then tested PTM at the protein level in a cellular model expressing RHO stably. While wild-type RHO was localized to the plasma membrane, mutant RHO was sequestrated in the cytoplasmic compartment. Using a flow-imaging methodology (ImageStreamX), we were able to precisely quantify this phenotype and showed that trans-splicing partially restored a correct localization of RHO in this cellular model. Thus, we constructed an AAV vector containing our best PTM to assess its activity in vivo in a humanized mouse model of rhodopsin mutation following subretinal injection. Using co-injection with an AAV-GFP and micro-dissection of the transduced part of the retina, we demonstrated that trans-splicing reached 30% at the RNA level, while it was not detectable in the untransduced part, or in animals that received a control PTM. This study demonstrates that trans-splicing may provide a therapeutic benefit in case of dominant mutations of the rhodopsin gene.
Phase I/II trial to establish the safety and preliminary efficacy of the combination of docetaxel, prednisone, and SOM 230 in metastatic castrate-resistant prostate cancer (CRPC).
Authors: H. M. Vankayala, L. K. Heilbrun, J. A. Fontana, U. N. Vaishampayan
Journal: Journal of Clinical Oncology (2011)

TPS185 Background: Docetaxel based therapy in metastatic castrate resistant prostate cancer (mCRPC) provides an improvement in progression free survival and overall survival. However cures are extremely unlikely. A mechanism of resistance to docetaxel is due to neuroendocrine differentiation of cells. Somatostatin (sst) receptor analogs have efficacy in neuroendocrine tumors. SOM-230 is a multitargeted sst receptor analog with five times the affinity as other sst receptor analogs and maybe able to overcome docetaxel resistance. Methods: This is an open label, phase I study with cohorts of 3-6 patients to determine the maximum tolerated dose (MTD). The MTD cohort will be expanded to 12 patients to assess efficacy. The primary objective is to establish the MTD level of SOM 230 in combination with docetaxel and prednisone. The secondary objectives are to evaluate the safety, tolerability and efficacy of the combination therapy. Eligible patients are chemotherapy naïve, with histologically confirmed mCRPC, objective progression or rising PSA despite androgen deprivation therapy and antiandrogen withdrawal. Eligible patients are >18yrs with ECOG performance status < 2 and have life expectancy >12 wks. Adequate hepatic, renal and bone marrow function is required. Patients will receive SOM-230 intramuscularly every 28 days in escalating doses. Docetaxel 75mg/m2 will be administered every 21 days along and prednisone at 5 mg twice daily orally. Correlatives: Circulating tumor cell counts, IGF-1 levels, and chromogranin A levels will be evaluated to assess proof of attacking the targets. Enrollment is due to start in may 2011. Conclusion: This study will determine the feasibility and tolerability of the combination of docetaxel, prednisone and SOM-230 in mCRPC and provide preliminary efficacy data. If favorable, then further investigation of this combination will be planned. Supported in part by Novartis Inc.
Telomere length, COPD and emphysema as risk factors for lung cancer
Authors: Juan P. de-Torres, Pablo Sanchez-Salcedo, Gorka Bastarrika, Ana B. Alcaide, Rubén Pío, María Jose Pajares, Arantza Campo, Juan Berto, Luis Montuenga, Maria del Mar Ocon, Carmen Monente, Bartolome R. Celli, Javier J. Zulueta
Journal: European Respiratory Journal (2016)

Telomeres are DNA–protein structures that protect chromosome ends from degradation and shorten progressively with each cell division [1]. Telomere length measured in peripheral leukocytes has been used to determine an individual's "biological age", and previous reports have demonstrated that patients with chronic obstructive pulmonary disease (COPD) and/or emphysema have shorter peripheral leukocyte telomere length [2–5]. The presence of COPD and emphysema are independent risk factors for the ultimate development of lung cancer [6, 7]. Leukocyte telomere length should be included among the independent risk factors associated with lung cancer <http://ow.ly/uzlg304Qpn6> J.P. de-Torres, G. Bastarrika, J.J. Zulueta, P. Sanchez-Salcedo, B.R. Celli (conception and design, analysis and interpretation, and drafting the manuscript for important intellectual content); A.B. Alcaide, J. Berto, A. Campo, M. del Mar Ocon, C. Monente, R. Pío, M.J. Pajares, L. Montuenga (analysis and interpretation).
A Distinct DNA Methylation Pattern Characterizes Childhood Acute Lymphoblastic Leukemia and Separates Patients Into Prognostic Groups,
Authors: Stefanie Göllner, Anika Witten, Monika Stoll, Wolfgang E. Berdel, Martin Schrappe, Gunnar Cario, Martin Stanulla, Carsten Müller-Tidow
Journal: Blood (2011)
169. Expression Studies of Ornithine Transcarbamylase in Liver Using Minicircle Vectors for Gene Therapy: Flag-Tagging of a Mitochondrial Enzyme and Stable Expression Under Control of an Endogenous Otc Promoter-Enhancer
Authors: Hiu Man Viecelli, Sereina Deplazes, Andrea Schlegel, Sharon Cunningham, Ian Alexander, Johannes Häberle, Beat Thöny
Journal: Molecular Therapy (2016)

Ornithine transcarbamylase deficiency (OTCD) is the most common inherited defect of the urea cycle resulting in severe hyperammonemia and death if left untreated. We are aiming at correcting OTCD using naked-DNA minicircle (MC) vector mediated gene therapy in the spfash mouse model with low residual OTC activity. A critical parameter is delivery to periportal hepatocytes where the urea cycle is located because of metabolic zonation in the liver. To distinguish between MC-born and endogenous OTC enzyme, we generated an expression cassette with an internally Flag-tagged OTC enzyme. Internal epitope tagging is required because of the N-terminal mitochondrial import sequence. A corresponding Flag-tag sequence was introduced C-terminally of the mitochondrial import signal. The tagged protein performed similar to its non-tagged OTC enzyme upon hydrodynamic tail vein injection for liver targeting in spfash mice, indicating that the tag neither interferes with the mitochondrial import nor the formation of a functional OTC trimer. Furthermore we generated an endogenous Otc promoter-enhancer construct, termed PmO1, for potential specific or "natural" OTC transgene expression. According to others, the promoter (672 bp) sequence derived from mouse Otc was not sufficient for liver specificity (Veres et al, J Biol Chem 261: 7588-7591, 1986) and requires a corresponding enhancer sequence (Nishiyori et al, J Biol Chem 269: 1323-1331, 1994). An enhancer sequence (232 bp) originating from rat Otc was used that contains several binding sites for liver-selective transcription factors (Murakami et al, Mol Cell Biol 10: 1180-1191, 1990). The resulting MC-vector expressing OTC from this PmO1 promoter-enhancer construct was delivered to spfash mice via hydrodynamic tail vein injection, resulting in similar enzymatic activity in whole liver extracts compared to wild-type mice. Vectors designed to express Flag-tagged OTC driven from a natural Otc promoter might be useful to study biodistribution and/or long-term stable expression after MC-based gene therapy for OTCD.
465. An Antibiotic-Free Strategy for Miniplasmid Production
Authors: Ram Shankar, Maurice Telaar, Karl Friehs, Martin Schleef, Erwin Flaschel
Journal: Molecular Therapy (2016)

The segregational stability of plasmids in a recombinant bioprocess is of extreme significance. Although this is commonly achieved by the selection pressure from antibiotics, their application for the production of therapeutic DNA for gene therapy or for DNA vaccines would be undesirable. Similarly, the presence of antibiotic-resistance genes in the final product would have to be avoided. In addition to the minicircle approach, a type of miniplasmid is able to fulfil the regulatory requirements. The gene tpiA is responsible for the connection of the glycerol metabolic pathway with the essential glycolytic pathway in Escherichia coli. The knockout of genomic tpiA rendered cells completely auxotrophic in minimal medium with glycerol as sole carbon source while allowing growth at a reduced rate with glucose or in complex medium. This was advantageous for optimizing antibiotic-free cloning and selection of recombinant plasmid. Complementation of the auxotrophy by plasmid-borne tpiA led to high segregational and structural plasmid stability, resulting in stable production of a model recombinant enzyme under antibiotic-free conditions in a continuous cultivation. Thus, the complementation of tpiA represents a significant alternative to antibiotics as a selection principle and is therefore of interest for the recombinant production of biotherapeutics in the form of miniplasmids.
Abstract 4395: Strategy to overcome inherent TRAIL-based therapeutic limitations
Authors: Yumin Oh, Maggie Swierczewska, Seulki Lee
Journal: Cancer Research (2015)

Abstract TNF-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent because of its selective ability to induce apoptosis in activated immune cells or cancer cells with minimal toxicity in normal cells. However, a short biological half-life and the development of inherent TRAIL resistance in a variety of cancer cells, including colorectal cancer cells (CRC), results in the poor performance of TRAIL as a chemotherapeutic agent. To overcome these limitation, we developed PEGylated TRAIL (PEG-TRAIL) to compensate the short half-life of TRAIL offering improved pharmacokinetics in non-human primates. In parallel, we found that pre-sensitization with doxorubicin resulted in the upregulated expression of the functional TRAIL receptor, DR5, in highly TRAIL-resistant HT-29 cells by Western blotting and qRT-PCR. Although neither individual treatment of doxorubicin nor TRAILPEG induced apoptosis, pre-sensitization with doxorubicin followed by PEG-TRAIL induced significant apoptosis through the activation of both extrinsic and intrinsic apoptotic pathways promoted by the cleavage of caspases 8, 9, and 3. DR5, but not DR4, was recruited to the TRAIL-induced Death-Inducing Signaling Complex (DISC) within HT-29 cells pre-sensitized with doxorubicin, suggesting a TRAIL/DR5 specific pathway in CRC. To maximize the therapeutic efficacy of the sensitizer in vivo, doxorubicin was encapsulated in tumor-homing hyaluronic acid nanoparticles (HAC/DOX). Confocal microscopy and HPLC analysis enabled us to observe the efficient uptake and biodistribution of HAC/DOX in tumor tissues from xenografted HT-29 cells. HAC/DOX effectively sensitized the xenograft tumor, bearing the highly TRAIL-resistant HT-29 cells to TRAIL-induced apoptosis through the upregulated expression of DR5 and activation of caspases. Our study suggests an innovative approach to overcome the inherent TRAIL-based therapeutic limitations by introducing long-acting TRAIL and a tumor-homing TRAIL sensitizer to induce cancer cell-specific apoptosis in TRAIL-resistant CRC, particularly in physiological conditions. Citation Format: Yumin Oh, Maggie Swierczewska, Seulki Lee. Strategy to overcome inherent TRAIL-based therapeutic limitations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4395. doi:10.1158/1538-7445.AM2015-4395
Screening, diagnosis, and management of patients with Fabry disease: conclusions from a “Kidney Disease: Improving Global Outcomes” (KDIGO) Controversies Conference
Authors: Raphael Schiffmann, Derralynn A. Hughes, Gabor E. Linthorst, Alberto Ortiz, Einar Svarstad, David G. Warnock, Michael L. West, Christoph Wanner, Daniel G. Bichet, Erik Ilsø Christensen, Ricardo Correa-Rotter, Perry M. Elliott, Sandro Feriozzi, Agnes B. Fogo, Dominique P. Germain, Carla E.M. Hollak, Robert J. Hopkin, Jack Johnson, Ilkka Kantola, Jeffrey B. Kopp, Jürgen Kröner, Aleš Linhart, Ana Maria Martins, Dietrich Matern, Atul B. Mehta, Renzo Mignani, Behzad Najafian, Ichiei Narita, Kathy Nicholls, Greg T. Obrador, João Paulo Oliveira, Antonio Pisani, Juan Politei, Uma Ramaswami, Markus Ries, Wim Terryn, Camilla Tøndel, Roser Torra, Bojan Vujkovac, Stephen Waldek, Jerry Walter
Journal: Kidney International (2016)

Patients with Fabry disease (FD) are at a high risk for developing chronic kidney disease and cardiovascular disease. The availability of specific but costly therapy has elevated the profile of this rare condition. This KDIGO conference addressed controversial areas in the diagnosis, screening, and management of FD, and included enzyme replacement therapy and nonspecific standard-of-care therapy for the various manifestations of FD. Despite marked advances in patient care and improved overall outlook, there is a need to better understand the pathogenesis of this glycosphingolipidosis and to determine the appropriate age to initiate therapy in all types of patients. The need to develop more effective specific therapies was also emphasized.
Qualitative and Quantitative Concordance of Minimal Residual Disease Data Assessed by Multicolor Flow Cytometry and PCR of Fusion Gene Transcripts In Childhood B-Cell Precursor Acute Lymphoblastic Leukemia
Authors: Alexander Popov, Grigory Tsaur, Tatiana Verzhbitskaya, Tatyana Riger, Yulia Yakovleva, Anna Ivanova, Alexander Druy, Egor Shorikov, Leonid Saveliev, Larisa Fechina
Journal: Blood (2010)