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Your search keyword '"Juan E. Hurtado"' showing total 4 results
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4 results on '"Juan E. Hurtado"'

1. Rapid, Single-Cell Analysis and Discovery of Vectored mRNA Transfection In Vivo with a loxP-Flanked tdTomato Reporter Mouse

Author

Kevin J. Kauffman, Matthias A. Oberli, J. Robert Dorkin, Juan E. Hurtado, James C. Kaczmarek, Shivani Bhadani, Jeff Wyckoff, Robert Langer, Ana Jaklenec, and Daniel G. Anderson

Subjects
mRNA, nanoparticles, reporter mouse, single-cell analysis, flow cytometry, Therapeutics. Pharmacology, RM1-950
Abstract

mRNA therapeutics hold promise for the treatment of diseases requiring intracellular protein expression and for use in genome editing systems, but mRNA must transfect the desired tissue and cell type to be efficacious. Nanoparticle vectors that deliver the mRNA are often evaluated using mRNA encoding for reporter genes such as firefly luciferase (FLuc); however, single-cell resolution of mRNA expression cannot generally be achieved with FLuc, and, thus, the transfected cell populations cannot be determined without additional steps or experiments. To more rapidly identify which types of cells an mRNA formulation transfects in vivo, we describe a Cre recombinase (Cre)-based system that permanently expresses fluorescent tdTomato protein in transfected cells of genetically modified mice. Following in vivo application of vectored Cre mRNA, it is possible to visualize successfully transfected cells via Cre-mediated tdTomato expression in bulk tissues and with single-cell resolution. Using this system, we identify previously unknown transfected cell types of an existing mRNA delivery vehicle in vivo and also develop a new mRNA formulation capable of transfecting lung endothelial cells. Importantly, the same formulations with mRNA encoding for fluorescent protein delivered to wild-type mice did not produce sufficient signal for any visualization in vivo, demonstrating the significantly improved sensitivity of our Cre-based system. We believe that the system described here may facilitate the identification and characterization of mRNA delivery vectors to new tissues and cell types.

Published
2018
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2. In vivo hypermutation and continuous evolution

Author

Rosana S. Molina, Gordon Rix, Amanuella A. Mengiste, Beatriz Álvarez, Daeje Seo, Haiqi Chen, Juan E. Hurtado, Qiong Zhang, Jorge Donato García-García, Zachary J. Heins, Patrick J. Almhjell, Frances H. Arnold, Ahmad S. Khalil, Andrew D. Hanson, John E. Dueber, David V. Schaffer, Fei Chen, Seokhee Kim, Luis Ángel Fernández, Matthew D. Shoulders, and Chang C. Liu

Subjects
General Medicine, Article, General Biochemistry, Genetics and Molecular Biology
Published
2022

3. Combinatorial drug discovery in nanoliter droplets

Author

Prianca Tawde, Anthony Kulesa, Jared Kehe, Paul C. Blainey, and Juan E. Hurtado

Subjects
0301 basic medicine, medicine.drug_class, Phenotypic screening, High-throughput screening, Antibiotics, Drug Evaluation, Preclinical, Microbial Sensitivity Tests, Computational biology, Pharmacology, Biology, 01 natural sciences, Chemical library, Small Molecule Libraries, 03 medical and health sciences, chemistry.chemical_compound, Vancomycin, Lab-On-A-Chip Devices, Drug Discovery, Escherichia coli, medicine, Combinatorial Chemistry Techniques, Nanotechnology, Multidisciplinary, 010405 organic chemistry, Drug discovery, Drug Synergism, Biological Sciences, Microarray Analysis, Chemical space, Anti-Bacterial Agents, Erythromycin, High-Throughput Screening Assays, 0104 chemical sciences, 030104 developmental biology, Synergy, chemistry, Infectious disease (medical specialty), Pseudomonas aeruginosa, Novobiocin
Abstract

Combinatorial drug treatment strategies perturb biological networks synergistically to achieve therapeutic effects and represent major opportunities to develop advanced treatments across a variety of human disease areas. However, the discovery of new combinatorial treatments is challenged by the sheer scale of combinatorial chemical space. Here we report a high-throughput system for nanoliter-scale phenotypic screening that stabilizes a chemical library in nanoliter droplet emulsions and automates the formulation of chemical combinationsen massusing parallel droplet processing. We apply this system to predict synergy between more than 4,000 investigational and approved drugs and a panel of 10 antibiotics againstE. coli, a model Gram-negative pathogen. We found a range of drugs not previously indicated for infectious disease that synergize with antibiotics. Our validated hits include drugs that synergize with the antibiotics vancomycin, erythromycin, and novobiocin, which are used against Gram-positive bacteria but are not effective by themselves to resolve Gram-negative infections.

Published
2018

4. Efficacy and immunogenicity of unmodified and pseudouridine-modified mRNA delivered systemically with lipid nanoparticles in vivo

Author

Kevin J. Kauffman, Michael W. Heartlein, Daniel G. Anderson, Matthew J. Webber, Piotr S. Kowalski, Juan E. Hurtado, Frank Derosa, Siddharth Jhunjhunwala, James C. Kaczmarek, Faryal F. Mir, Jung H. Yang, Institute for Medical Engineering and Science, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Chemical Engineering, Koch Institute for Integrative Cancer Research at MIT, Kauffman, Kevin John, Mir, Faryal, Jhunjhunwala, Siddharth, Kaczmarek, James Cliff, Hurtado, Juan E., Yang, Jung H, Webber, Matthew, Kowalski, Piotr S, and Anderson, Daniel Griffith

Subjects
0301 basic medicine, Surface Properties, Biophysics, Gene Expression, Bioengineering, Pharmacology, Biology, Transfection, Pseudouridine, Article, Biomaterials, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Immune system, In vivo, Extracellular, Animals, Humans, Myeloid Cells, RNA, Messenger, Particle Size, Messenger RNA, Innate immune system, Immunogenicity, Gene Transfer Techniques, Molecular biology, Lipids, Uridine, Immunity, Innate, Mice, Inbred C57BL, 030104 developmental biology, chemistry, Liver, Mechanics of Materials, 030220 oncology & carcinogenesis, Ceramics and Composites, Cytokines, Nanoparticles, Female, HeLa Cells
Abstract

mRNA has broad potential for treating diseases requiring protein expression. However, mRNA can also induce an immune response with associated toxicity. Replacement of uridine bases with pseudouridine has been postulated to modulate both mRNA immunogenicity and potency. Here, we explore the immune response and activity of lipid nanoparticle-formulated unmodified and pseudouridine-modified mRNAs administered systemically in vivo. Pseudouridine modification to mRNA had no significant effect on lipid nanoparticle physical properties, protein expression in vivo, or mRNA immunogenicity compared to unmodified mRNA when delivered systemically with liver-targeting lipid nanoparticles, but reduced in vitro transfection levels. Indicators of a transient, extracellular innate immune response to mRNA were observed, including neutrophilia, myeloid cell activation, and up-regulation of four serum cytokines. This study provides insight into the immune responses to mRNA lipid nanoparticles, and suggests that pseudouridine modifications may be unnecessary for therapeutic application of mRNA in the liver. Keywords: mRNA, Base modification, Pseudouridine, Lipid nanoparticle, In vivo, Immmune response, Shire Pharmaceuticals

Published
2016

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