scholarly journals CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting

Gene Therapy ◽  
2015 ◽  
Vol 23 (1) ◽  
pp. 108-112 ◽  
Author(s):  
D G Courtney ◽  
J E Moore ◽  
S D Atkinson ◽  
E Maurizi ◽  
E H A Allen ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Specific Targeting

Targeted chemical nucleases have a wide range of untapped applications in biological fields, including gene therapy

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Metal Complex ◽  
Dna Cleavage ◽  
Therapeutic Potential ◽  
Chemical Reactivity ◽  
Major Groove ◽  
Sequence Recognition ◽  
Wide Range ◽  
Chemical Nucleases

A feasible alternative to state-of-the-art enzymatic nucleases was created by regulating the cleavage activity of metal complexes using (covalent or non-covalent) homing agents. Targeted AMNs, unlike enzymatic nucleases, break DNA by an oxidative mechanism and can therefore permanently knock off genes. Compared to larger enzymatic nucleases, the modest size of the metal complex may aid cellular transfection. Furthermore, the painstaking construction of the sequence-specific probe permits a metal complex to be directed to dsDNA's minor or major groove. To direct the chemical reactivity of several small-molecule compounds to dsDNA's minor groove, covalently bonded polyamide samples were used. PNA and DNA were also used to construct antisense and antigen hybrids, with Watson–Crick or Hoogsteen base pairing with major groove nucleobases giving sequence recognition. Click chemistry created chimeric AMN-TFOs with desirable focused effects and negligible off-target cleavage. Clip-Phen-modified TFOs, 230 polypyridyl-modified TFOs, 232 and intercalating phenanthrene-modified TFOs are three contemporary instances of copper AMN–TFOs. All three systems have distinct advantages in maintaining the desired 2:1 phenthroline/copper ratio for DNA cleavage (clip-Phen TFOs), caging the copper center and facilitating efficient ROS-mediated strand scission (polypyridyl-modified TFO) and improving triplex stability (polypyridyl-modified TFO) (phenanthrene-TFOs). Cerium (IV)/EDTA complexes, recently shown to bind and hydrolytically cleave ssDNA/dsDNA junctions and used in conjunction with PNA to successfully introduce genome changes in vitro and in vivo, are another important class of targeted chemical nucleases. The chemical reactivity and wide flexibility of metal complex design, combined with their coupling to sequence specific samples for directed applications, show that these compounds have a wide range of untapped applications in biological fields such as chemotherapy, protein engineering, DNA footprinting, and gene editing. Parallel advancements in cell and tissue targeting will be essential to maximise their therapeutic potential, either by using specific ligands or creating new targeting modalities.

LB0002 A FIRST IN CLASS THERAPEUTIC NANOPARTICLE FOR SPECIFIC TARGETING OF ANTI-CITRULLINATED PROTEIN ANTIBODY AMELIORATES SERUM TRANSFER AND COLLAGEN INDUCED ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 212-212
Author(s):  
S. Khatri ◽  
J. Hansen ◽  
M. H. Clausen ◽  
T. W. Kragstrup ◽  
S. C. Hung ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Near Infrared ◽  
Infrared Imaging ◽  
Specific Targeting ◽  
Serum Transfer ◽  
Nanoparticle Formulation ◽  
Citrullinated Protein ◽  
Citrullinated Peptide

Background:Rheumatoid arthritis (RA) is an immune mediated inflammatory disease with autoimmune features, including antibodies to citrullinated proteins and peptides (ACPAs). Several in vitro studies have suggested a pathogenic role of ACPAs in RA. However, in vivo proof of this concept has been hampered by the lack of therapeutic strategies to reduce or deplete ACPA in serum and synovial fluid. Previously, we constructed a chitosan-hyaluronic acid nanoparticle formulation with the ability to use neutrophil recruitment as a delivery mechanism to inflamed joints. Specifically, nanoparticles got phagocytosed and then released to synovial fluid upon death of the short-lived neutrophilsObjectives:We hypothesized that reducing ACPA levels would have a therapeutic effect by blocking cytokine production. In this study, we prepared and tested a series of therapeutic nanoparticles for specific targeting of ACPA in synovial fluid.Methods:Nanoparticles were prepared by the microdroplet method and then decorated with synthetic cyclic citrullinated peptide aptamer PEP2, PEG/hexanoic acid and fluorophore (Cy5.5). Nanoparticles were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM) and high-performance liquid chromatography (HPLC). Nanoparticles were then used in a series of in vitro assays, including cell uptake with flow cytometry (FACS) detection, and in vivo studies including disease activity scores, cytokine measurements and near-infrared imaging.Results:We screened a series of citrullinated peptide epitopes and identified a fibrinogen-derived 21-amino-acid-long citrullinated peptide showing high selectivity toward autoantibodies in RA samples. We incorporated this aptamer in the chitosan-hyaluronic acid nanoparticle formulation previously described. Average nanoparticle size was 230 nm ± 10 nm by DLS and SEM; z potential was -0.0012. Purity by HPLC was over 95%. Attachment efficiency of the aptamer was 92% by HPLC. FACS study showed selective uptake of Cy5.5 labelled aptamer-nanoparticle conjugates by neutrophils in the concentration range 0.5-4 nM. Similar to previous studies,1there was no apparent immunogenicity for this nanoparticle formulation measured by cytokine secretion from human peripheral blood leukocytes. In vivo, over 50% reduction of disease activity was achieved in three weeks treatment using as little as 1 nM drug candidate (dosed every 48 hours) in the collagen-induced (CIA) mouse model of RA (N=30; p<0.001 for treated vs placebo). Same was observed in the serum transfer model (N=10). The aptamer-nanoparticle conjugate significantly reduced IL-6 and TNFα levels in the mouse sera (p<0.01). The effects were not inferior to tocilizumab treated controls (N=30). To confirm mode of action, we applied Cy5.5-labelled aptamer-nanoparticles in the collagen-induced mouse model (N=10) and analyzed the resulting uptake by near-infrared imaging. We confirmed over 6-fold higher signal accumulation in inflamed vs healthy joints (p<0.01), which strongly supports the fact that the aptamer is highly specific to the inflammatory process.Conclusion:Overall, we have designed a first-in-class therapeutic nanoparticle drug for specific targeting of anti-citrullinated protein antibodies. The marked effect of this nanoparticle observed in vivo holds promise for targeting ACPAs as a therapeutic option in RA.References:[1]Khatri S, Hansen J, Mendes AC, Chronakis IS, Hung S-C, Mellins ED, Astakhova K. Bioconjug Chem. 2019 Oct 16;30(10):2584–259Disclosure of Interests:Sangita Khatri: None declared, Jonas Hansen: None declared, Mads Hartvig Clausen Shareholder of: iBio Tech ApS, Tue Wenzel Kragstrup Shareholder of: iBio Tech ApS, Consultant of: Bristol-Myers Squibb, Speakers bureau: TWK has engaged in educational activities talking about immunology in rheumatic diseases receiving speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, and UCB., Shu-Chen Hung: None declared, Elisabeth Mellins: None declared, Kira Astakhova: None declared

scholarly journals A dual-targeting ruthenium nanodrug that inhibits primary tumor growth and lung metastasis via the PARP/ATM pathway

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yu Lu ◽  
Di Zhu ◽  
Lin Gui ◽  
Yuanming Li ◽  
Wenjing Wang ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Lung Metastasis ◽  
Dna Cleavage ◽  
Primary Tumor ◽  
Cell Cycle Progression ◽  
Chloride Ions ◽  
Lewis Lung Cancer ◽  
Dual Targeting

Abstract Background Many studies have found that ruthenium complexes possess unique biochemical characteristics and inhibit tumor growth or metastasis. Results Here, we report the novel dual-targeting ruthenium candidate 2b, which has both antitumor and antimetastatic properties and targets tumor sites through the enhanced permeability and retention (EPR) effect and transferrin/transferrin receptor (TF/TFR) interaction. The candidate 2b is composed of ruthenium-complexed carboline acid and four chloride ions. In vitro, 2b triggered DNA cleavage and thus blocked cell cycle progression and induced apoptosis via the PARP/ATM pathway. In vivo,2b inhibited not only Lewis lung cancer (LLC) tumor growth but also lung metastasis. We detected apoptosis and decreased CD31 expression in tumor tissues, and ruthenium accumulated in the primary tumor tissue of C57BL/6 mice implanted with LLC cells. Conclusions Thus, we conclude that 2b targets tumors, inhibits tumor growth and prevents lung metastasis.

scholarly journals Folate-Targeted Transgenic Activity of Dendrimer Functionalized Selenium Nanoparticles In Vitro

2020 ◽  
Vol 21 (19) ◽  
pp. 7177
Author(s):  
Nikita Simone Pillay ◽  
Aliscia Daniels ◽  
Moganavelli Singh
Keyword(s):  
Transgene Expression ◽  
In Vitro Cytotoxicity ◽  
Selenium Nanoparticles ◽  
In Vivo Studies ◽  
Cellular Interaction ◽  
Nucleic Acid Binding ◽  
Specific Targeting ◽  
Cancer Types

Current chemotherapeutic drugs, although effective, lack cell-specific targeting, instigate adverse side effects in healthy tissue, exhibit unfavourable bio-circulation and can generate drug-resistant cancers. The synergistic use of nanotechnology and gene therapy, using nanoparticles (NPs) for therapeutic gene delivery to cancer cells is hereby proposed. This includes the benefit of cell-specific targeting and exploitation of receptors overexpressed in specific cancer types. The aim of this study was to formulate dendrimer-functionalized selenium nanoparticles (PAMAM-SeNPs) containing the targeting moiety, folic acid (FA), for delivery of pCMV-Luc-DNA (pDNA) in vitro. These NPs and their gene-loaded nanocomplexes were physicochemically and morphologically characterized. Nucleic acid-binding, compaction and pDNA protection were assessed, followed by cell-based in vitro cytotoxicity, transgene expression and apoptotic assays. Nanocomplexes possessed favourable sizes (<150 nm) and ζ-potentials (>25 mV), crucial for cellular interaction, and protected the pDNA from degradation in an in vivo simulation. PAMAM-SeNP nanocomplexes exhibited higher cell viability (>85%) compared to selenium-free nanocomplexes (approximately 75%), confirming the important role of selenium in these nanocomplexes. FA-conjugated PAMAM-SeNPs displayed higher overall transgene expression (HeLa cells) compared to their non-targeting counterparts, suggesting enhanced receptor-mediated cellular uptake. Overall, our results bode well for the use of these nano-delivery vehicles in future in vivo studies.

scholarly journals ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITIES OF RUBUS FRUTICOSUS AND ZIZYPHUS VULGARIS METHANOL EXTRACTS

2017 ◽  
Vol 9 (2) ◽  
pp. 69
Author(s):  
Asma Meziti ◽  
Hamama Bouriche ◽  
Meziti Hichem ◽  
Seoussen Kada ◽  
Abderrahmane Senator ◽  
...  
Keyword(s):  
Oral Administration ◽  
Antioxidant Capacity ◽  
Vascular Permeability ◽  
Dna Cleavage ◽  
Total Antioxidant Capacity ◽  
Rubus Fruticosus ◽  
Total Antioxidant ◽  
Anti Inflammatory

<p class="Default"><strong>Objective</strong>:<strong> </strong>This report is an attempt to study the phenolic composition of <em>Rubus fruticosus </em>(RFE) and<em> Zizyphus vulgaris (</em>ZVE<em>) </em>methanol<em> </em>extracts<em> and </em>evaluate their antioxidant and anti-inflammatory effects <em>in-vitro</em> and <em>in-vivo</em>.</p><p><strong>Methods: </strong>Total phenolic and total flavonoids contents of extracts were determined by spectrophotometric methods. Phenolic compounds were identified by<strong> </strong>HPLC-TOF/MS. The antioxidant activities were evaluated <em>in vitro</em> using DPPH, ABTS and FRAP assays. The effect of RFE and ZVE<em> </em>on DNA cleavage induced by H<sub>2</sub>O<sub>2</sub> UV-photolysis was also investigated. The antioxidant effect of RFE and ZVE was tested <em>in vivo</em> using the blood total antioxidant capacity test in mice. On the other hand, the anti-inflammatory activity was assessed <em>in vivo</em> using two models of acute inflammation ear edema and vascular permeability.</p><p><strong>Results: </strong>The phytochemical analysis of these extracts showed that RFE possesses higher polyphenolic and flavonoid content than ZVE. in the same way RFE exerted the highest antioxidant capacity with IC<sub> 50 </sub>value of 14 µg/ml in DPPH assay, 1.58 mmol of Trolox E/mg extract and 3.39 of mmol FesO<sub>4</sub>/mg extract in ABTS, and FRAP assay respectively. The studied extracts showed a concentration-dependent protective effect on DNA cleavage induced by H<sub>2</sub>O<sub>2</sub> UV-photolysis. The daily oral administration of 200 mg/kg of RFE or ZVE during three weeks showed an improvement of the blood total antioxidant capacity; the HT<sub>50</sub> values were151.45 min and 146.72 min for the groups treated with RFE and ZVE, respectively <em>versus</em> 122.5 min for the control group. The topical application of 2 mg/ear of RFE inhibited the croton oil-induced ear edema by 75.72%, while the inhibition exerted by ZVE was 64.24%. These inhibitions were higher than that of indomethacin, used as a reference. Moreover, the oral administration of 400 mg/kg of RFE inhibited significantly (33.57%) acetic acid induced vascular permeability in mice. However, this effect was lower than this of indomethacin. The inhibition effect exerted by ZVE was not significant.</p><p><strong>Conclusion</strong><strong>:</strong><strong> </strong>The results obtained in this investigation showed that RFE possesses strong antioxidant and anti-inflammatory potential in comparison with ZVE, which may be attributed to the presence of polyphenolic phytoconstituents.</p>

scholarly journals A novel 18F-labeled clickable substrate for targeted imaging of SNAP-tag expressing cells by PET in vivo

2021 ◽  
Author(s):  
Dominic Depke ◽  
Christian Paul Konken ◽  
Lukas Rösner ◽  
Sven Hermann ◽  
Michael Schäfers ◽  
...  
Keyword(s):  
High Potential ◽  
Targeted Imaging ◽  
Covalent Labeling ◽  
Specific Targeting

Bioorthogonal covalent labeling with self-labeling enzymes like SNAP-tag bears a high potential for specific targeting of cells for imaging in vitro and in vivo. To this end, fluorescent SNAP substrates...

scholarly journals Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation

Blood ◽  
2009 ◽  
Vol 113 (13) ◽  
pp. 2965-2975 ◽  
Author(s):  
William Giblin ◽  
Monalisa Chatterji ◽  
Gerwin Westfield ◽  
Tehmina Masud ◽  
Brian Theisen ◽  
...  
Keyword(s):  
Immune System ◽  
Dna Cleavage ◽  
Severe Combined Immunodeficiency ◽  
Double Strand Breaks ◽  
Mutant Form ◽  
Dna Rearrangements ◽  
Strand Breaks ◽  
Immune System Dysfunction

Abstract The RAG1/2 endonuclease initiates programmed DNA rearrangements in progenitor lymphocytes by generating double-strand breaks at specific recombination signal sequences. This process, known as V(D)J recombination, assembles the vastly diverse antigen receptor genes from numerous V, D, and J coding segments. In vitro biochemical and cellular transfection studies suggest that RAG1/2 may also play postcleavage roles by forming complexes with the recombining ends to facilitate DNA end processing and ligation. In the current study, we examine the in vivo consequences of a mutant form of RAG1, RAG1-S723C, that is proficient for DNA cleavage, yet exhibits defects in postcleavage complex formation and end joining in vitro. We generated a knockin mouse model harboring the RAG1-S723C hypomorphic mutation and examined the immune system in this fully in vivo setting. RAG1-S723C homozygous mice exhibit impaired lymphocyte development and decreased V(D)J rearrangements. Distinct from RAG nullizygosity, the RAG1-S723C hypomorph results in aberrant DNA double-strand breaks within rearranging loci. RAG1-S723C also predisposes to thymic lymphomas associated with chromosomal translocations in a p53 mutant background, and heterozygosity for the mutant allele accelerates age-associated immune system dysfunction. Thus, our study provides in vivo evidence that implicates aberrant RAG1/2 activity in lymphoid tumor development and premature immunosenescence.

Glycyrrhetinic acid-induced apoptosis in thymocytes: impact of 11β-hydroxysteroid dehydrogenase inhibition

1999 ◽  
Vol 277 (4) ◽  
pp. E624-E630 ◽  
Author(s):  
Hiroshi Horigome ◽  
Atsushi Horigome ◽  
Masato Homma ◽  
Toshihiko Hirano ◽  
Kitaro Oka
Keyword(s):  
Dna Cleavage ◽  
Flow Cytometric Analysis ◽  
Intraperitoneal Administration ◽  
Hydroxysteroid Dehydrogenase ◽  
Glycyrrhetinic Acid ◽  
Immunocytochemical Staining ◽  
Double Positive ◽  
Corticosterone Concentration

It has been proposed that glycyrrhetinic acid (GA) enhances endogenous glucocorticoid (GC) action by suppressing the metabolism of the steroid. We show here that marked involution of the thymus occurred within 24 h of a single intraperitoneal administration of GA in mice. Thymocytes from mice treated with GA exhibited DNA cleavage and mitochondrial transmembrane potential disruption, as demonstrated with agarose gel electrophoresis and flow cytometric analysis. Immunocytochemical staining revealed that CD4+CD8+double positive cells markedly decreased after GA treatment. In contrast to GA in vivo, GA in vitro did not induce apoptosis of cultured thymocytes. These findings suggest that the apoptosis-inducing effect of GA on thymocytes is due to its indirect action. Because GA has been known to inhibit 11β-hydroxysteroid dehydrogenase (11β-HSD), we measured the enzyme activity in major organs and endogenous corticosterone concentration after GA treatment. The results showed a significant decrease of 11β-HSD activity ( P < 0.0001) and an increase in serum corticosterone concentration ( P< 0.005). We concluded that the inhibition of hepatic 11β-HSD activity by GA has a serious effect on GC metabolism, which results in a significant elevation of systemic GC levels. Apoptosis of thymocytes occurred as a consequence of the elevation in the level of endogenous corticosterone.

scholarly journals Hin Recombinase Mutants Functionally Disrupted in Interactions with Fis

2001 ◽  
Vol 183 (1) ◽  
pp. 28-35 ◽  
Author(s):  
Oliver Z. Nanassy ◽  
Kelly T. Hughes
Keyword(s):  
Dna Binding ◽  
Dna Cleavage ◽  
Genetic Screen ◽  
Recombination Reaction ◽  
Genetic Classification ◽  
Hin Recombinase

ABSTRACT A previous genetic screen was designed to separate Hin recombinase mutants into distinct classes based on the stage in the recombination reaction at which they are blocked (O. Nanassy, Zoltan, and K. T. Hughes, Genetics 149:1649–1663, 1998). One class of DNA binding-proficient, recombination-deficient mutants was predicted by genetic classification to be defective in the step prior to invertasome formation. Based on the genetic criteria, mutants from this class were also inferred to be defective in interactions with Fis. In order to understand how the genetic classification relates to individual biochemical steps in the recombination reaction these mutants, R123Q, T124I, and A126T, were purified and characterized for DNA cleavage and recombination activities. Both the T124I and A126T mutants were partially active, whereas the R123Q mutant was inactive. The A126T mutant was not as defective for recombination as the T124I allele and could be partially rescued for recombination both in vivo and in vitro by increasing the concentration of Fis protein. Rescue of the A126T allele required the Fis protein to be DNA binding proficient. A model for a postsynaptic role for Fis in the inversion reaction is presented.

An insight into the mechanism of inhibition of unusual bi-subunit topoisomerase I from Leishmania donovani by 3,3′-di-indolylmethane, a novel DNA topoisomerase I poison with a strong binding affinity to the enzyme

2007 ◽  
Vol 409 (2) ◽  
pp. 611-622 ◽  
Author(s):  
Amit Roy ◽  
Benu Brata Das ◽  
Agneyo Ganguly ◽  
Somdeb Bose Dasgupta ◽  
Neeta V. M. Khalkho ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Dna Cleavage ◽  
Topoisomerase I ◽  
Leishmania Donovani ◽  
Wide Spectrum ◽  
Free Enzyme ◽  
Rational Drug Design ◽  
Binary Complex

DIM (3,3′-di-indolylmethane), an abundant dietary component of cruciferous vegetables, exhibits a wide spectrum of pharmacological properties. In the present study, we show that DIM is a potent inhibitor of Leishmania donovani topoisomerase I with an IC50 of 1.2 μM. Equilibrium dialysis shows that DIM binds strongly to the free enzyme with a binding constant of 9.73×10−9 M. The binding affinity of DIM to the small subunit is 8.6-fold more than that of the large subunit of unusual LdTOP1LS (bi-subunit L. donovani topoisomerase I). DIM stabilizes topoisomerase I–DNA cleavage complexes in vitro and also in vivo. Like CPT (camptothecin), DIM inhibits the religation step when the drug was added to preformed topoisomerase I–DNA binary complex. Hence, DIM is similar to CPT with respect to its ability to form the topoisomerase I-mediated ‘cleavable complexes’ in vitro and in vivo. But unlike CPT, DIM interacts with both free enzyme and substrate DNA. Therefore DIM is a non-competitive class I inhibitor of topoisomerase I. DIM also inhibits the relaxation activity of the CPT-resistant mutant enzyme LdTOP1Δ39LS (N-terminal deletion of amino acids 1–39 of LdTOP1LS). The IC50 values of DIM in simultaneous and enzyme pre-incubation relaxation assays were 3.6 and 2.9 μM respectively, which are higher than that of wild-type topoisomerase I (LdTOP1LS), indicating that the affinity of DIM to LdTOP1Δ39LS is less than that for LdTOP1LS. This is the first report on DIM as an L. donovani topoisomerase I poison. Our study illuminates a new mode of action of enzyme inhibition by DIM that might be exploited for rational drug design in human leishmaniasis.

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