Genetic Engineering & Biotechnology News

AUG 2018

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Page 17 of 33

16 | AUGUST 2018 | Genetic Engineering & Biotechnology News | Brian P. Dranka, Ph.D., and Luke Dimasi Immune cells are some of the most dynamic cells in the body. They can up- and downregulate cellular processes to meet the critical and multifaceted functions of surveillance, activa- tion, proliferation, cytokine production, antibody secretion, and pathogen clearance. Even though these functions can be analyzed and characterized with a vast array of methods (such as flow cytometry, gene expression profiling, and mea- surement of cytokine production), laboratories still struggle to interrogate the dynamic and rapid nature of immune cell biology in real time. To capture functional shifts in real time, laboratories are adopting another technique: the monitoring of energy me- tabolism. This technique not only complements established methods, it also enables a better understanding of the im- mediate to early functions of immune cells. Changes in cellular metabolism reflect metabolic repro- gramming—a novel and unique marker—and occur on the order of minutes to allow changes in cell function. These metabolic changes may not only contribute to cell function changes, they may also be sufficient to cause these changes. Moreover, these metabolic changes provide a valuable set of pathway targets for modulating immune cell response, func- tion, and fate. The need to study the real-time kinetic responses is espe- cially relevant in neutrophil biology. Neutrophils are phago- cytic cells that represent the main antimicrobial defense of the innate immune response, and like most immune cells, neutrophils quickly ramp up glycolysis to meet their rapidly changing cellular energetic and metabolic demands. Glucose metabolism is also important to sustain the pen- tose phosphate pathway that generates nicotinamide adenine dinucleotide phosphate (NADPH), one of the substrates of the NADPH oxidase (NOX2) enzyme and necessary for neutrophil extracellular trap release. Upon stimulation, the membrane-associated NOX2 is activated, resulting in a rapid and powerful oxidative burst, during which a large amount of oxygen is consumed to generate superoxide and concomitant other reactive oxygen species (ROSs). Genera- tion of ROSs is critical for effective antimicrobial immunity and inflammatory response. Established assays to detect and quantify oxidative bursts are indirect and based on fluorescent/luminescent detection of ROSs derived from superoxide anion formation. Despite good sensitivity, these methods are not specific, are prone to artifacts, are sensitive to the compartmentalization of the probe, and do not allow proper understanding of the dura- tion and inactivation phase of the response, since the oxi- dized probe irreversibly accumulates during the assay. Real-Time Monitoring and Modulation of Neutrophil Activation he continues. "Thus, every targeting event results in the killing of that cell." In contrast, Cas9 produces clean double-strand breaks that are repaired by DNA repair mechanisms in bacteria, which ultimately allow for the sur- vival of the cell. At Locus, the hope is to create therapeu- tics that could replace antibiotics. To put this hope to the test, the company is moving to- ward a clinical trial with the help of approxi- mately $19 million in funding that was se- cured in 2017. Currently, Locus researchers are using CRISPR to develop a new drug for complicated urinary tract infections caused by Escherichia coli bacteria, with a spe- cific focus on the multidrug-resistant E. coli strains. Two other drugs following close be- hind include targeted therapies for Pseudo- monas aeruginosa and Clostridium difficile. eGenesis The Boston-based eGenesis is a CRISPR startup co-founded by another genome engi- neering pioneer, George M. Church, Ph.D., a geneticist, molecular engineer, and chemist who holds senior academic posts at Harvard Medical School, MIT, and the Wyss Insti- tute. By leveraging gene editing technologies, eGenesis is working to regularize xenotrans- plantation in medicine. The company's am- bition, the eGenesis website declares, is the delivery of "safe and effective human trans- plantable cells, tissues, and organs to the hundreds of thousands of patients worldwide who are in dire need." In March 2017, the company raised ap- proximately $38 million and devoted part of its funding toward developing CRISPR-Cas9 technology that can be used to resolve a key xenotransplantation challenge: the inactiva- tion of porcine endogenous retroviruses (PER- Vs). Because they can infect humans, PERVs have, to date, complicated the development of safer transplant-friendly pig organs. "By leveraging CRISPR technology, eGen- esis has the tools to tackle the cross-species viral transmission concern in xenotransplan- tation that was difficult to address before," Luhan Yang, Ph.D., chief scientific officer and co-founder of eGenesis, tells GEN. Last year, eGenesis scientists reported that they not only used CRISPR-Cas9 to inactivate all of the PERVs in a porcine primary cell line, they also generated PERV-inactivated pigs via somatic cell nuclear transfer. These achievements, according to a paper written by eGenesis scientists and published in Science, opens the door to a safer source of organs and tissues for pig-to-human xeno- transplantation. "The team has created more than 30 pigs that have been born PERV-free," she adds. "Today, these piglets are likely the most advanced genome-modified animals liv- ing on Earth." CRISPR Startups Continued from page 15 OMICS Assay Tutorial Agilent Technologies' Quantitative Analysis Method Incorporates a Cellular Bioenergetics Platform Figure 1. This XF neutrophil activation assay kinetic trace shows that ox ygen consumption is an early measure of neutrophil ac tivation. Notice how the oxygen consumption rate (OCR) changes in the presence of mitochondrial inhibitors rotenone and antimycin A (Rot/AA). Initially, 0.5 µM of each inhibitor was injected to eliminate the mitochondrial contribution to OCR. This was followed 24 min later by an injection of an activator, 100 ng/mL phorbol myristate acetate (PMA), or vehicle control (black arrows). Figure 2. These XF neutrophil activation kinetic traces show real-time OCR data that were obtained when different doses of the activator PMA were administered. (Human peripheral blood neutrophils were plated at 4 × 10 4 per well of XF96 cell culture microplates in the presence of Rot/AA.) Such data may lead to a better understanding of the mechanism and time-course of neutrophil activation, and contribute to the development of novel agents that maintain activation at a level that allows suppression of infection while preventing inflammatory responses. Figure 3. In neutrophils, an oxidative burst requires glycolysis. An XF neutrophil activation kinetic trace of OCR (top) and an XF neutrophil activation kinetic trace of proton efflux rate (PER) (bottom) in the presence of the inhibitor 2-deoxy- d -glucose (2-DG). Notice that 2-DG (or vehicle), Rot/AA, and PMA (or vehicle) were serially admistered (black arrows). 2-DG (intial): 50 mM; Rot/AA (6 min later): 0.5 µM; PMA (24 min later): 100 ng/mL. Human peripheral blood neutrophils were plated at 4 × 10 4 per well.

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