Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs

2005 ◽  
Vol 23 (8) ◽  
pp. 1002-1007 ◽  
Author(s):  
David V Morrissey ◽  
Jennifer A Lockridge ◽  
Lucinda Shaw ◽  
Karin Blanchard ◽  
Kristi Jensen ◽  
...  
Keyword(s):  
Chemically Modified

scholarly journals Chemically Modified Synthetic microRNA-205 Inhibits the Growth of Melanoma Cells In Vitro and In Vivo

2013 ◽  
Vol 21 (6) ◽  
pp. 1204-1211 ◽  
Author(s):  
Shunsuke Noguchi ◽  
Junya Iwasaki ◽  
Minami Kumazaki ◽  
Takashi Mori ◽  
Kohji Maruo ◽  
...  
Keyword(s):  
Melanoma Cells ◽  
Chemically Modified

scholarly journals Functional Analysis of Coordinated Cleavage in V(D)J Recombination

1998 ◽  
Vol 18 (8) ◽  
pp. 4679-4688 ◽  
Author(s):  
Deok Ryong Kim ◽  
Marjorie A. Oettinger
Keyword(s):  
Functional Analysis ◽  
Divalent Cation ◽  
Chromosomal Translocations ◽  
Cleavage Reaction ◽  
Chemically Modified ◽  
Double Stranded Dna ◽  
Cleavage Complex ◽  
Helical Turn

ABSTRACT V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn2+, but cleavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.

scholarly journals Combining enyne metathesis with long-established organic transformations: a powerful strategy for the sustainable synthesis of bioactive molecules

2020 ◽  
Vol 16 ◽  
pp. 738-755
Author(s):  
Valerian Dragutan ◽  
Ileana Dragutan ◽  
Albert Demonceau ◽  
Lionel Delaude
Keyword(s):  
Therapeutic Agents ◽  
Ruthenium Catalysts ◽  
Diels Alder ◽  
Bioactive Molecules ◽  
Enyne Metathesis ◽  
Organic Transformations ◽  
Chemically Modified ◽  
Key Steps ◽  
Powerful Strategy

This account surveys the current progress on the application of intra- and intermolecular enyne metathesis as main key steps in the synthesis of challenging structural motifs and stereochemistries found in bioactive compounds. Special emphasis is placed on ruthenium catalysts as promoters of enyne metathesis to build the desired 1,3-dienic units. The advantageous association of this approach with name reactions like Grignard, Wittig, Diels–Alder, Suzuki–Miyaura, Heck cross-coupling, etc. is illustrated. Examples unveil the generality of such tandem reactions in providing not only the intricate structures of known, in vivo effective substances but also for designing chemically modified analogs as valid alternatives for further therapeutic agents.

scholarly journals Protein - Quinone Interaction: In Vitro Induction of Indirect Antiglobulin Reactions With Methyldopa

Blood ◽  
1974 ◽  
Vol 43 (1) ◽  
pp. 85-97 ◽  
Author(s):  
Arlan J. Gottlieb ◽  
Harold A. Wurzel
Keyword(s):  
Gamma Globulin ◽  
Red Cell ◽  
Prolonged Incubation ◽  
Chemically Modified ◽  
Latex Beads ◽  
Structural Analogues ◽  
In Vitro Induction ◽  
Equilibrium Centrifugation

Abstract Methyldopa-treated gamma globulin can be demonstrated serologically on either the red cell surface or on latex beads by the indirect antiglobulin reaction. The development of a positive antiglobulin reaction was related to methyldopa concentration and the length and temperature of incubation of methyldopa with protein and could be partially inhibited by the addition of albumin to the incubation mixtures. After more prolonged incubation, antiglobulin positivity also developed with plasma-treated with methyldopa. 14C-methyldopa was covalently bound to gamma globulin. Aggregation of gamma globulin following treatment with methyldopa could be demonstrated by both sedimentation velocity and molecular weight determinations employing low-speed equilibrium centrifugation. Protein aggregation was a function of time, temperature, and methyldopa concentration. Detectability by the antiglobulin reaction, the darkening noted in solutions to which methyldopa or hydroquinone had been added, as well as the aggregation of protein was inhibited by a reducing agent which prevented formation of a quinone from the hydroquinone. Some of the immunologically atypical features of the sensitization of red cells by methyldopa or its structural analogues are explicable by the adherence, in vivo, of chemically modified, nonantibody gamma globulin which renders the red cell directly antiglobulin positive.

scholarly journals Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 817
Author(s):  
Tsuyoshi Yamamoto ◽  
Yahiro Mukai ◽  
Fumito Wada ◽  
Chisato Terada ◽  
Yukina Kayaba ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Ex Vivo ◽  
The Body ◽  
Locked Nucleic Acid ◽  
Imaging Studies ◽  
Phosphodiester Bond ◽  
Chemically Modified ◽  
Ex Vivo Imaging ◽  
Mirna Targeting

The development of clinically relevant anti-microRNA antisense oligonucleotides (anti-miRNA ASOs) remains a major challenge. One promising configuration of anti-miRNA ASOs called “tiny LNA (tiny Locked Nucleic Acid)” is an unusually small (~8-mer), highly chemically modified anti-miRNA ASO with high activity and specificity. Within this platform, we achieved a great enhancement of the in vivo activity of miRNA-122-targeting tiny LNA by developing a series of N-acetylgalactosamine (GalNAc)-conjugated tiny LNAs. Specifically, the median effective dose (ED50) of the most potent construct, tL-5G3, was estimated to be ~12 nmol/kg, which is ~300–500 times more potent than the original unconjugated tiny LNA. Through in vivo/ex vivo imaging studies, we have confirmed that the major advantage of GalNAc over tiny LNAs can be ascribed to the improvement of their originally poor pharmacokinetics. We also showed that the GalNAc ligand should be introduced into its 5′ terminus rather than its 3′ end via a biolabile phosphodiester bond. This result suggests that tiny LNA can unexpectedly be recognized by endogenous nucleases and is required to be digested to liberate the parent tiny LNA at an appropriate time in the body. We believe that our strategy will pave the way for the clinical application of miRNA-targeting small ASO therapy.

scholarly journals Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Amal Alex ◽  
Valentina Piano ◽  
Soumitra Polley ◽  
Marchel Stuiver ◽  
Stephanie Voss ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Mammalian Cells ◽  
Cellular Localization ◽  
Mitotic Checkpoint ◽  
Transferase Activity ◽  
Checkpoint Signaling ◽  
Chemically Modified ◽  
Promising Tool

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.

scholarly journals Prime editing primarily induces undesired outcomes in mice

2020 ◽  
Author(s):  
Tomomi Aida ◽  
Jonathan J. Wilde ◽  
Lixin Yang ◽  
Yuanyuan Hou ◽  
Mengqi Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Frequency ◽  
Mouse Embryos ◽  
Biomedical Science ◽  
New Approach ◽  
Chemically Modified ◽  
Highly Efficient ◽  
Large Deletions

SummaryGenome editing has transformed biomedical science, but is still unpredictable and often induces undesired outcomes. Prime editing (PE) is a promising new approach due to its proposed flexibility and ability to avoid unwanted indels. Here, we show highly efficient PE-mediated genome editing in mammalian zygotes. Utilizing chemically modified guideRNAs, PE efficiently introduced 10 targeted modifications including substitutions, deletions, and insertions across 6 genes in mouse embryos. However, we unexpectedly observed a high frequency of undesired outcomes such as large deletions and found that these occurred more often than pure intended edits across all of the edits/genes. We show that undesired outcomes result from the double-nicking PE3 strategy, but that omission of the second nick largely ablates PE function. However, sequential double-nicking with PE3b, which is only applicable to a fraction of edits, eliminated undesired outcomes. Overall, our findings demonstrate the promising potential of PE for predictable, flexible, and highly efficient in vivo genome editing, but highlight the need for improved variations of PE before it is ready for widespread use.

Overcoming analytical challenges to generate data critical to understanding lipid nanoparticle-delivered modified mRNA biodistribution

Bioanalysis ◽  
2019 ◽  
Vol 11 (21) ◽  
pp. 1993-2001 ◽  
Author(s):  
Glen Hawthorne ◽  
Neil Henderson ◽  
Mikko Hölttä ◽  
Sima Khan ◽  
Johnny Lindqvist ◽  
...  
Keyword(s):  
Lipid Nanoparticles ◽  
Sample Collection ◽  
Safety And Efficacy ◽  
Chemically Modified ◽  
Novel Approach ◽  
Mrna Therapeutics ◽  
Successful Analysis ◽  
Processing Steps ◽  
Novel Delivery Systems

Aim: Chemically modified mRNA offers a novel approach to treat disease. Due to susceptibility to extracellular nucleases in vivo, dosed modified mRNA therapeutics can benefit from encapsulation within novel delivery systems, such as lipid nanoparticles (LNPs). To understand the holistic effect of dosing LNP-encapsulated modified mRNA therapeutics can require bioanalysis of several components including the mRNA, protein and LNP. Methodology: These components can require bespoke preanalytical strategies to preserve analyte integrity to achieve successful analysis. Here we describe the sample collection, processing steps and bioanalytical technologies that can be used to overcome these challenges. Discussion: Understanding the biodistribution and holistic effects of the different components allow the pharmaceutical industry to evaluate safety and efficacy of modified mRNA therapeutics.

Sign in / Sign up

Export Citation Format

Share Document