8 results on '"Devin Pillis"'
Search Results
2. NRAS
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Elisa, Boscolo, Patricia, Pastura, Sandra, Schrenk, Jillian, Goines, Rachael, Kang, Devin, Pillis, Punam, Malik, and Timothy D, Le Cras
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Mice ,Vascular Malformations ,Mutation ,Animals ,Endothelial Cells ,Humans ,Membrane Proteins ,Mice, Nude ,Article ,GTP Phosphohydrolases - Abstract
Somatic mutations in NRAS drive the pathogenesis of melanoma and other cancers but their role in vascular anomalies and specifically human endothelial cells is unclear. The goals of this study were to determine whether the somatic-activating NRAS(Q61R) mutation in human endothelial cells induces abnormal angiogenesis and to develop in vitro and in vivo models to identify disease-causing pathways and test inhibitors. Here, we used mutant NRAS(Q61R) and wild-type NRAS (NRAS(WT)) expressing human endothelial cells in in vitro and in vivo angiogenesis models. These studies demonstrated that expression of NRAS(Q61R) in human endothelial cells caused a shift to an abnormal spindle-shaped morphology, increased proliferation, and migration. NRAS(Q61R) endothelial cells had increased phosphorylation of ERK compared to NRAS(WT) cells indicating hyperactivation of MAPK/ERK pathways. NRAS(Q61R) mutant endothelial cells generated abnormal enlarged vascular channels in a 3D fibrin gel model and in vivo, in xenografts in nude mice. These studies demonstrate that NRAS(Q61R) can drive abnormal angiogenesis in human endothelial cells. Treatment with MAP kinase inhibitor U0126 prevented the change to a spindle-shaped morphology in NRAS(Q61R) endothelial cells, whereas mTOR inhibitor rapamycin did not.
- Published
- 2021
3. CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites
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Devin Pillis, Fan Zhang, Michael A. Goodman, Rajeswari Jayavaradhan, Paul R. Andreassen, Yue Zhang, and Punam Malik
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CRISPR-Cas9 genome editing ,0301 basic medicine ,DNA End-Joining Repair ,DNA Repair ,Molecular biology ,DNA repair ,Science ,Mutant ,General Physics and Astronomy ,02 engineering and technology ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,03 medical and health sciences ,Genome editing ,CRISPR-Associated Protein 9 ,Humans ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,DNA Breaks, Double-Stranded ,lcsh:Science ,Gene Editing ,Multidisciplinary ,Cas9 ,fungi ,Recombinational DNA Repair ,General Chemistry ,DNA Repair Pathway ,021001 nanoscience & nanotechnology ,Fusion protein ,Cell biology ,Mutagenesis, Insertional ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Genetic engineering ,embryonic structures ,lcsh:Q ,CRISPR-Cas Systems ,Tumor Suppressor p53-Binding Protein 1 ,0210 nano-technology ,Biotechnology - Abstract
Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition., Global inhibition of NHEJ factors has been one strategy to improve CRISPR-Cas9 mediated HDR. Here the authors fuse a dominant-negative mutant of 53BP1 to Cas9 to enhance HDR frequency, reduce NHEJ specifically at the Cas9 cut sites, and reduce the toxicity associated with global NHEJ inhibition.
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- 2019
4. A Versatile Tool for the Quantification of CRISPR/Cas9-Induced Genome Editing Events in Human Hematopoietic Cell Lines and Hematopoietic Stem/Progenitor Cells
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Devin Pillis, Rajeswari Jayavaradhan, and Punam Malik
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DNA Repair ,Computational biology ,Biology ,Genome engineering ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Genome editing ,Structural Biology ,CRISPR ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,Progenitor cell ,Molecular Biology ,030304 developmental biology ,Gene Editing ,0303 health sciences ,Hematopoietic cell ,Cas9 ,Hematopoietic Stem Cells ,body regions ,Haematopoiesis ,chemistry ,CRISPR-Cas Systems ,030217 neurology & neurosurgery ,DNA ,RNA, Guide, Kinetoplastida - Abstract
The efficient site-specific DNA double-strand breaks (DSB) created by CRISPR/Cas9 has revolutionized genome engineering and has great potential for editing hematopoietic stem/progenitor cells (HSPCs). However, detailed understanding of the variables that influence choice of DNA-DSB repair (DDR) pathways by HSPC is required for therapeutic levels of editing in these clinically relevant cells. We developed a hematopoietic-reporter system that rapidly quantifies the three major DDR pathways utilized at the individual DSB created by CRISPR/Cas9-NHEJ, MMEJ, and HDR-and show its applicability in evaluating the different DDR outcomes utilized by human hematopoietic cell lines and primary human HSPC.
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- 2018
5. Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential
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Johannes Christiaan Maria van der Loo, Mehdi Keddache, Paritha Arumugam, Michael A. Goodman, Dennis D. Hickstein, Danielle Lynn, Phillip J. Dexheimer, Anastacia Loberg, Mohammed Nasimuzzaman, Devin Pillis, David W. Russell, Punam Malik, and Thomas R. Bauer
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0301 basic medicine ,viruses ,Immunology ,Genetic Vectors ,Insulator (genetics) ,Microbiology ,Proto-Oncogene Mas ,Virus ,Viral vector ,03 medical and health sciences ,Mice ,Gene Delivery ,Retrovirus ,Transduction, Genetic ,Virology ,CRISPR ,Animals ,Transgenes ,Enhancer ,Cells, Cultured ,Gammaretrovirus ,Adaptor Proteins, Signal Transducing ,biology ,Mutagenicity Tests ,Terminal Repeat Sequences ,Genetic Therapy ,LIM Domain Proteins ,biology.organism_classification ,Hematopoietic Stem Cells ,Long terminal repeat ,Mutagenesis, Insertional ,030104 developmental biology ,Insect Science ,Spumavirus ,Insulator Elements ,CRISPR-Cas Systems - Abstract
Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in “enhancerless” self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required. IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy.
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- 2017
6. Gene Therapy for Sickle Cell Anemia Using a Modified Gamma Globin Lentivirus Vector and Reduced Intensity Conditioning Transplant Shows Promising Correction of the Disease Phenotype
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Stella M. Davies, Archana Shreshta, Johannes C.M. Van der Loo, Jennifer Knight-Madden, Theodosia A. Kalfa, Frederic D. Bushman, Monika Asnani, Michael Grimley, Punam Malik, Omar Niss, Elke Grassman, Carolyn Lutzko, Lilith Reeves, Parinda A. Mehta, Amy Shova, Catherine E. Terrell, Sydney Felker, Loberg Anastacia, Charles T. Quinn, Karen Kalinyak, Scott Witting, Little Courtney, Sharat Chandra, Devin Pillis, and Diana Nordling
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0301 basic medicine ,medicine.medical_specialty ,Neutrophil Engraftment ,business.industry ,Anemia ,Plerixafor ,Thalassemia ,Immunology ,Genetic transfer ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Gastroenterology ,Sickle cell anemia ,Transplantation ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,Internal medicine ,medicine ,Bone marrow ,business ,medicine.drug - Abstract
Background: Genetic transfer of an anti-sickling β87-globin lentiviral vector (LV) into hematopoietic stem cells (HSC) followed by myeloablative transplant has cured one child with sickle cell anemia (SCA) (NEJM 2017), although it was not successful in 7 subsequent adult SCA patients, and modifications to intensify ablative conditioning, improve HSC dose, gene transfer are underway (Blood 130 Suppl 1: 527, 2017). Based upon our preclinical data (Blood 2009), we embarked upon a Reduced Intensity Conditioning (RIC) Phase I/II Pilot Study on Gene Transfer in Patients with SCA with a modified γ-Globin LV (NCT02186418), hypothesizing this approach will be safe, feasible and efficacious; Moreover, RIC will have significantly less toxicity, costs, and be implementable in many transplant centers, including those in some of the resource-poor countries, where supportive therapies for myeloablative transplants are unavailable, and where majority of SCA patients exist. Methods: Adult patients with severe SCA deemed eligible were transfused/erythrocytapheresed prior to HSC collection and transfused for 6 months post-transplant (PT) to Hb>10g/dl and HbS~30%. CD34+ HSC were collected via bone marrow harvest (BMH) and/or plerixafor mobilized Peripheral Blood Stem Collection (PBSC), selected for CD34+ cells and transduced. Patients received a single dose of IV melphalan (140mg/m2 BSA) 36hr prior to infusion of γ-globin modified (GM)-HSC. Patients were monitored for adverse events (AE), engraftment, vector copy number (VCN), modified HbF (HbF*) expression and clinical features of SCA. Results: Two SCA patients (35yo and 25yo) with HbS-β0 thalassemia genotype were treated. CD34+ HSC were collected via multiple BMH (P1) and BMH+PBSC (P2). Follow up data are available for 6 and 12mo on P1 and P2. P1 received 1x106 CD34+ cells/kgbw [vector copy number (VCN) 0.22], and P2 received 6.9x106 CD34+ cells/kgbw [VCN 0.46]. Time to neutrophil engraftment (ANC ≥ 500) was day 9 and 7 post-transplant (PT) in P1 and P2, respectively, and time to Plt recovery (Plt>50K) was day 14 PT in both. Patients included in this trial had severe disease and continued to have pre-existing chronic pain requiring significant opiates; hence ~80% of the AEs were pain events; other AEs were anticipated transient laboratory AEs associated with melphalan. Following GM-HSC infusion, both patients showed a progressive rise in HbF* (a point mutation in the γ-globin LV allows distinction from endogenous HbF by HPLC) starting from day 30 PT. Since patients had transfused HbA containing RBCs in the initial 6 months, HbF*/(HbF*+HbS) was calculated, and was 20% and 21% in P1 and P2 at day 180 PT and VCN 0.2-0.4, detected in all lineages. Integration site analysis, performed on the infused products (Day 0), at day 30 PT on P1 and P2, and on day 180 PT on P1 demonstrated highly polyclonal pattern of integration. At 1 yr PT, P1 had 20% HbF* (2.1g/dl HbF*, total Hb 10.6) with a stable VCN of 0.2-0.4 in multiple lineages in bone marrow and peripheral blood. The baseline Hb of P1 was 7.5-8.5g/dL prior to transplant. In the preceding 2 years prior to transplant, both patients were admitted for pain crises/acute chest >5-6 times/yr, and had chronic pain requiring chronic opiates. Chronic pain persisted for 4-5 months PT in P1, after which P1 has not required IV opiates, negligible oral opiates and has had no hospital visits/admissions with acute sickle events. P2 has required decreasing amounts of oral opiates for chronic back pain. Conclusions: Early results from 2 SCA adults treated with a modified γ-globin LV modified autologous HSC following RIC transplant showed excellent safety, feasibility, with minimal post-transplant toxicity, rapid count recovery, and sustained stable genetically modified cells in peripheral blood and bone marrow. The first patient shows significant clinical amelioration of the SCA phenotype at 1 year PT, with 20% vector-derived HbF (HbF*) that has caused amelioration of anemia, near elimination of chronic pain and absence of acute sickle events. The second patient, although still early post-transplant shows a similar HbF* trajectory. Additional study data will demonstrate whether this level of HbF* will provide consistent clinical benefit to patients with severe SCA. These early results, especially following a RIC transpant, are extremely promising; and if sustained, will provide a 'transportable' safe and feasible gene therapy for SCA. Disclosures Malik: CSL Behring: Patents & Royalties. Quinn:Global Blood Therapeutics: Research Funding; Silver Lake Research Corporation: Research Funding; Amgen: Research Funding.
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- 2018
7. Placenta growth factor augments airway hyperresponsiveness via leukotrienes and IL-13
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Vijay K. Kalra, Timothy D. Le Cras, Nancy Ratner, Shiva Kumar Shanmukhappa, Eric B. Brandt, Gurjit K. Khurana Hershey, Devin Pillis, Peter Carmeliet, Bart Jonck, Anastacia Loberg, Tilat A. Rizvi, Paritha Arumugam, Swati Tiwari, Marsha Wills-Karp, Marthe Sandrine Eiymo Mwa Mpollo, Mark Lindsey, and Punam Malik
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medicine.medical_specialty ,Leukotrienes ,Inflammation ,Anemia, Sickle Cell ,Pregnancy Proteins ,Mice ,Th2 Cells ,Internal medicine ,medicine ,Eosinophilia ,Animals ,Hydroxyurea ,Respiratory system ,Asthma ,STAT6 ,Placenta Growth Factor ,Mice, Knockout ,Leukotriene ,Interleukin-13 ,business.industry ,General Medicine ,Zileuton ,respiratory system ,medicine.disease ,respiratory tract diseases ,Disease Models, Animal ,Endocrinology ,Immunology ,Interleukin 13 ,medicine.symptom ,business ,medicine.drug ,Research Article - Abstract
Airway hyperresponsiveness (AHR) affects 55%-77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the mechanisms underlying the high AHR prevalence in a hemoglobinopathy remain unknown. We hypothesized that placenta growth factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates AHR. In allergen-exposed mice, loss of Plgf dampened AHR, reduced inflammation and eosinophilia, and decreased expression of the Th2 cytokine IL-13 and the leukotriene-synthesizing enzymes 5-lipoxygenase and leukotriene-C4-synthase. Plgf-/- mice treated with leukotrienes phenocopied the WT response to allergen exposure; conversely, anti-PlGF Ab administration in WT animals blunted the AHR. Notably, Th2-mediated STAT6 activation further increased PlGF expression from lung epithelium, eosinophils, and macrophages, creating a PlGF/leukotriene/Th2-response positive feedback loop. Similarly, we found that the Th2 response in asthma patients is associated with increased expression of PlGF and its downstream genes in respiratory epithelial cells. In an SCD mouse model, we observed increased AHR and higher leukotriene levels that were abrogated by anti-PlGF Ab or the 5-lipoxygenase inhibitor zileuton. Overall, our findings indicate that PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. These data also suggest that zileuton and anti-PlGF Ab could be promising therapies to reduce pulmonary morbidity in SCD.
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- 2014
8. Foamy Virus Backbone Has Insulator Properties Which Remarkably Reduce Its Genotoxicity Potential
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Johannes C.M. Van der Loo, Dennis D. Hickstein, Mehdi Keddache, Punam Malik, Paritha Arumugam, David W. Russell, Devin Pillis, Thomas R. Bauer, Michael A. Goodman, and Danielle Lynn
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LMO2 ,Transgene ,Immunology ,Promoter ,Cell Biology ,Hematology ,Transfection ,Biology ,biology.organism_classification ,Biochemistry ,Molecular biology ,Retrovirus ,CTCF ,hemic and lymphatic diseases ,Enhancer ,Gene - Abstract
Strong viral enhancers in γ-retrovirus vectors (GV) have caused cellular proto-oncogene activation and leukemia in gene therapy trials, necessitating the use of cellular promoters in enhancer-less integrating vectors. However, data is now emerging that inadequate transgene expression from cellular promoters may limit successful correction. Vectors derived from foamy virus (FV), a nonpathogenic retrovirus, have a higher preference for non-genic integrations than GV/lentiviral vectors (LV), and may be less genotoxic. We constructed GV, LV and FV driven either by the spleen focus forming virus (SFFV) or the murine stem cell virus (MSCV) enhancer/promoters, all driving eGFP expression, and compared their relative genotoxicity using an in vitro immortalization assay on primary hematopoietic stem/progenitor cells (HSPC). In this assay, integration near a protooncogene/gene promoting cell proliferation results in quantifiable HSPC immortalization. Strong viral enhancer/promoters from SFFV or MSCV in FV caused a remarkably low immortalization of HSPC compared to analogous LV or GV: compared to the immortalization frequency of HSPC with the SFFV-GV in this assay, SFFV-LV and MSCV-LV had 12- and 14-fold lower immortalization frequency, while the SFFV-FV and MSCV-FV showed a 155- and 414-fold lower immortalization frequency, respectively. Immortalized clones had multiple (3-10) integrated copies. Integration site analysis of FV immortalized clones revealed a majority of integrants in non-gene regions; those in genic regions targeted cell proliferation or proto-oncogenes, as previously reported. FV has been previously reported to have 2-fold higher insertions in non-genic regions and higher, but nearly half the propensity to target promoters compared to GV. However, this remarkably reduced genotoxicity with FV was not explained by the integration pattern. We therefore hypothesized that FV backbone may contain sequences that have an enhancer blocking/insulator effect. Studies on chromatin insulators have shown that enhancer-blocking property of insulators is mediated via binding of CTCF to its consensus sequences within the insulator. Indeed, an in silico analysis for CCCTC-binding factor (CTCF) binding sites in the vector backbone sequences showed more predicted CTCF binding sites in the FV than in GV or LV (26, 8, and 6, respectively). To functionally validate the enhancer-blocking effect of the FV backbone and ensure that only effects of the vector backbone would be measured, without the confounding influence of integration site or the enhancer/promoter/transgene, we inserted SFFV-GV, SFFV-LV and SFFV-FV into a clinically relevant proto-oncogene, LMO2, using CRISPR/Cas9, and assessed LMO2 expression. LMO2 upregulation has previously resulted in leukemias in the X-linked severe combined immune-deficiency and Wiscott-Aldrich syndrome (WAS) GV-mediated gene transfer trials; notably SFFV-GV was used in the WAS trial and caused leukemias in 8 of 10 patients from insertional oncogenesis. HeLa cells were transfected with the proviral donor plasmids and the guide-RNA/spCas9 plasmids and GFP+ cells sorted and cloned. Nearly all clones derived had one intact LMO2 allele, while the other alleles had GV/LV/FV proviral insertions. We next assessed LMO2 mRNA and protein expression in these clones. We found a minimal increase in LMO2 mRNA expression with SFFV-FV, in sharp contrast to significantly increased LMO2 expression with SFFV-GV and SFFV-LV by qRT-PCR (Figure 1A). Overall, the SFFV enhancer in GV demonstrated the greatest fold-increase in LMO2 expression (median increase of 280+/-23-fold over unmodified HeLa cells), followed by the SFFV enhancer in LV (median 200+/-27-fold increase). However, the same SFFV enhancer in FV only showed a 45+/-7-fold median increase in expression. Western blot analysis for LMO2 protein expression in three clones for each vector showed no detectable LMO2 expression in SFFV-FV clones, which was similar to baseline in mock (non-targeted) HeLa cells (Figure 1B). However, significantly higher LMO2 protein was detectable in GV and LV clones. Hence, the remarkably low genotoxic potential of FV, even those carrying strong viral enhancers appears to be, in large part, from an insulator property of FV sequences, making FV ideal for situations where high transgene expression, necessitating strong enhancers is required for a therapeutic effect. Disclosures No relevant conflicts of interest to declare.
- Published
- 2016
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