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Researchers Pioneer New Method to Edit Genes in Human Cells

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SAN FRANCISCO, CA—January 5, 2021—Over the past decade, the CRISPR genome-editing system has revolutionized molecular biology, giving scientists the ability to alter genes inside living cells for research or medical applications. Now, researchers at Gladstone Institutes have fine-tuned an additional system for more efficient gene editing, using molecules called retrons.

SAN FRANCISCO, CA—January 5, 2021—Over the past decade, the CRISPR genome-editing system has revolutionized molecular biology, giving scientists the ability to alter genes inside living cells for research or medical applications. Now, researchers at Gladstone Institutes have fine-tuned an additional system for more efficient gene editing, using molecules called retrons.

Retrons, the group reported in the journal Nature Chemical Biology, can be optimized for efficiency and used to edit genes in a variety of cell types, from fungi to human cells.

“This work really solidifies retrons as a platform that can be used across organisms,” says Gladstone Assistant Investigator Seth Shipman, PhD, senior author of the new study. “We can make precise modifications to genes more easily, quickly, and efficiently than with current approaches.”

A One-Stop Shop for Gene Editing

Most current gene-editing technologies based on the CRISPR system involve cutting a section of DNA out of a cell’s genome, and then introducing new genetic material called “template DNA” to replace it. As the cell repairs the places where an existing gene was cut, the template DNA is integrated.

That template DNA is normally produced in the lab and then introduced to cells from the outside. The protein that cuts the cell’s genome—called Cas9—is delivered separately. Neither Cas9 nor the template DNA penetrate every cell, limiting the efficiency of CRISPR gene editing.

Retrons, however, act like DNA factories, producing abundant copies of template DNA from inside cells. Moreover, retrons can be delivered along with the rest of the CRISPR components so that cells get all the material needed for gene editing simultaneously—the genetic codes for template DNA, Cas9, and molecules that help researchers track the edits that have been made.

“This means we only have to introduce one element to each cell,” says Santiago Lopez, a graduate student in the Shipman Lab and first author of the new paper. “That significantly simplifies the process and opens the door for new types of experiments.”

Re-Engineering Retrons

Both retrons and CRISPR originate from bacteria; both are defense mechanisms that bacteria use to alter DNA in response to infections. After the advent of CRISPR genome editing, in which the CRISPR system was co-opted to selectively target genes in other cell types, some researchers began probing whether retrons could be used to supply the templates for precise gene editing. However, the roles of different sections of the retron’s structure in its function—and how to tweak those sections to improve retrons—has been unknown.

“The retron system evolved to help defend bacteria,” says Shipman, who is also an assistant professor of bioengineering and therapeutic sciences at UC San Francisco (UCSF). “But we wanted to change it from what it does normally to what we want it to do—produce templates for gene editing.”

In the new study, Shipman’s group engineered E. coli retrons to create hundreds of new variants. They tested each new variant and discovered a series of changes that, together, led to an 8-to-10-fold increase in how much template DNA was eventually produced by the retron in E. coli cells.

Next, the researchers tested the new re-engineered retron system in the fungus Saccharomyces cerevisiae (Baker’s yeast) and in cultured human cells, and they found that this optimized system worked in all cases. This was the first demonstration of retrons’ use in human cells and their portability across cell types.

Since the team could now fine-tune exactly how much template DNA the retrons produced, they were also able to show that when the retrons produce high levels of template DNA, this boosts gene editing efficiency.

“Our study demonstrates for the first time that the more template DNA we can produce, the better the genome editing,” says Shipman. “Better and more precise editing ultimately means more effective and safer genomic medicines and more advanced fundamental research.”

Taking Tools from Bacteria

Retrons, Shipman says, are immediately useful as a research tool for editing genes in different cell types in the lab. While the platform isn’t yet ready for use in humans, it also holds the potential to help edit genes for therapeutic purposes—for example by repairing gene mutations that cause disease.

Since different bacteria contain different retrons, his group also plans to explore whether other retron variations have benefits over the E. coli retron they optimized in this study.

“We’re taking a general approach in which we’re mining parts that we find in bacteria and domesticating them for our own use,” says Shipman. “This has already been incredibly fruitful for developing new tools, but I think we’re only just beginning to reap the benefits of applying these tools in biotechnology.”

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About the Study

The paper “Precise genome editing across kingdoms of life using retron-derived DNA” was published in the journal Nature Chemical Biology on December 23, 2021. Other authors are Kate Crawford, Sierra Lear, and Santi Bhattarai-Kline of Gladstone Institutes.

The work was supported by the Simons Foundation Autism Research Initiative, the Pew Scholars Program in the Biomedical Sciences, the Sandler Program for Breakthrough Biomedical Research at UCSF, the National Institutes of Health (1DP2GM140917-01), the L.K. Whittier Foundation, the National Science Foundation (graduate research fellowships 2019247827 and 2034836), a Berkeley Fellowship for Graduate Study, and the Discovery Fellows Program at UCSF.

About Gladstone Institutes

To ensure our work does the greatest good, Gladstone Institutes focuses on conditions with profound medical, economic, and social impact—unsolved diseases. Gladstone is an independent, nonprofit life science research organization that uses visionary science and technology to overcome disease. It has an academic affiliation with the University of California, San Francisco.

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Source: bioengineer.org

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Are Researchers One Step Closer to Developing the Theory of Impulse Circuits?

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Computers play an important role in many aspects of life today. Digital computers are the most widely used, while quantum computers are well known. However, the least known computers are the so-called Stochastic Pulse Computers. Their work is based on highly parallel logical operations between trains of electrical pulses, where the pulses occur at random times, as in neurons, the nerve cells in the brains of humans and mammals.

Computers play an important role in many aspects of life today. Digital computers are the most widely used, while quantum computers are well known. However, the least known computers are the so-called Stochastic Pulse Computers. Their work is based on highly parallel logical operations between trains of electrical pulses, where the pulses occur at random times, as in neurons, the nerve cells in the brains of humans and mammals.

The main motivation for the growing interest in research on RPC computers over the past decade is the hope that they could solve faster and with less energy consumption tasks that are normally easy for living beings, but difficult for digital computers, such as instantaneous responses to stimuli, pattern recognition, robustness to errors and damage in the system, learning, and autonomy.

In a recent study published in Scientific Reports, researchers from the Croatian Centre of Excellence for Advanced Materials and Sensors, Dr Mario Stipčević of the Ruđer Bošković Institute (RBI) and Mateja Batelić, a student at the Faculty of Science at the University of Zagreb (FS), Croatia, describe new or improved versions of RPC circuits that use quantum randomness for the first time, but also go a significant step further and lay the first foundation for RPC circuit theory.

Namely, while circuits for processing information in a digital computer can be assembled from logic circuits as building blocks based on the well-known Boolean theory, a similar theory for RPC circuits does not yet exist. Therefore, the synthesis of circuits for an RPC is limited to trial and error through experimentation or simulation.

‘’The central part of our paper is the formulation and proof of the so-called entropy budget theorem, which can be used to easily verify whether a given mathematical (or logical) operation can be performed or “calculated” by any physical circuit, and if so, how much excess entropy must be available to a circuit in order to perform the given operation.

In this paper, we demonstrate the theorem using several examples of mathematical operations. Perhaps the most interesting proof is the existence of a deterministic half-sum circuit (a + b) / 2. However, this circuit is not yet known, and finding it is a challenge for further research,” says Mario Stipčević, head of the Laboratory of Photonics and Quantum Optics at the Ruđer Bošković Institute.

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PSMA PET Validates EAU Classification System to Determine Risk of Prostate Cancer Recurrence

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Reston, VA (January 20, 2022)—New research has confirmed the accuracy of the novel European Association of Urology (EAU) risk classification system that groups prostate cancer patients based on their risk of recurrence. Prostate-specific membrane antigen (PSMA) PET imaging of men with prostate cancer validated the EAU groupings and provided insights that could further refine risk assessment for patients. This study was published in the January issue of The Journal of Nuclear Medicine.

Credit: Justin Ferdinandus, Wolfgang P. Fendler, Andrea Farolfi, et al.

Reston, VA (January 20, 2022)—New research has confirmed the accuracy of the novel European Association of Urology (EAU) risk classification system that groups prostate cancer patients based on their risk of recurrence. Prostate-specific membrane antigen (PSMA) PET imaging of men with prostate cancer validated the EAU groupings and provided insights that could further refine risk assessment for patients. This study was published in the January issue of The Journal of Nuclear Medicine.

The diagnostic workup of prostate cancer has changed rapidly over the past few years. Recently, the EAU introduced a clinical system separating patients with rising PSA values after first-line therapy (prostate surgery or radiation) into groups of those with high risk and those with low risk for development of metastases. Shortly after this, the U.S. Food and Drug Administration approved 68Ga-PSMA-11 as the first PET drug to target the PSMA for men with prostate cancer.

“Given the growing availability of PSMA-directed PET imaging, our study sought to assess disease in patients based on the EAU classifications while using PSMA PET to identify subgroups of patients, such as those with undetectable, locoregional or distant metastatic disease,” said Justin Ferdinandus, MD, nuclear medicine physician at University Hospital in Essen, Germany.

The multicenter, international study analyzed PSMA PET scans of nearly 2,000 patients with prostate cancer and rising PSA levels. Patterns of disease spread on PSMA PET imaging were used to classify prostate cancer patients into both low- and high-risk groups. High-risk groups were found to have higher rates of metastatic disease on PSMA PET compared to low-risk groups. However, PSMA PET also found metastatic disease in low-risk and no disease in high-risk patients.

“Our study underscores the utility of the EAU risk groups to determine risk of metastasis in biochemically recurrent prostate cancer. But not every high-risk patient has metastases and not every low-risk patient has locoregional or no disease,” said Wolfgang Fendler, MD, nuclear medicine physician at University Hospital in Essen.

He continued, “The ultimate aim of imaging is to provide the right treatment for each patient. As evidenced in this research, the accuracy of PSMA PET is essential to improve stratification and potentially outcomes both in low-risk and high-risk settings.” 

The authors of “PSMA PET validates higher rates of metastatic disease for European Association of Urology Biochemical Recurrence Risk Groups: an international multicenter study” include Justin Ferdinandus, Department of Nuclear Medicine, University of Duisburg-Essen and German Cancer Consortium (DKTK)-University Hospital Essen, Essen, Germany; Wolfgang P. Fendler and Ken Hermann, Department of Nuclear Medicine, University of Duisburg-Essen and German Cancer Consortium (DKTK)-University Hospital Essen, Essen, Germany, and Ahmanson Translational Imaging Division, Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California; Andrea Farolfi, Department of Nuclear Medicine, University of Duisburg-Essen and German Cancer Consortium (DKTK)-University Hospital Essen, Essen, Germany, and Division of Nuclear Medicine, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; Samuel Washington, Department of Urology, University of California San Francisco, San Francisco, California, and Department of Epidemiology and Statistics, University of California San Francisco, San Francisco, California; Osama Mohamad, Department of Radiation Oncology, University of California San Francisco, San Francisco, California; Miguel H. Pampaloni and Thomas A. Hope, Department of Radiology and Biomedical Imaging, University of California San Francisco, San Francisco, California; Peter J.H. Scott, Melissa Rodnick, Benjamin L. Viglianti and Morand Piert, Department of Radiology, University of Michigan, Ann Arbor, Michigan; Matthias Eiber, Department of Nuclear Medicine, Klinikum rechts der Isar, Technical University Munich, Munich, Germany; and Johannes Czernin, Wesley R. Armstrong and Jeremie Calais, Ahmanson Translational Imaging Division, Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California.

Visit JNM’s new website for the latest research, and follow our new Twitter and Facebook pages @JournalofNucMed.

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Please visit the SNMMI Media Center for more information about molecular imaging and precision imaging. To schedule an interview with the researchers, please contact Rebecca Maxey at (703) 652-6772 or [email protected].
 

About JNM and the Society of Nuclear Medicine and Molecular Imaging
The Journal of Nuclear Medicine (JNM) is the world’s leading nuclear medicine, molecular imaging and theranostics journal, accessed more than 13 million times each year by practitioners around the globe, providing them with the information they need to advance this rapidly expanding field. Current and past issues of The Journal of Nuclear Medicine can be found online at http://jnm.snmjournals.org.

JNM is published by the Society of Nuclear Medicine and Molecular Imaging (SNMMI), an international scientific and medical organization dedicated to advancing nuclear medicine and molecular imaging—precision medicine that allows diagnosis and treatment to be tailored to individual patients in order to achieve the best possible outcomes. For more information, visit www.snmmi.org.

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UCLA Researchers Develop Novel Microscopic Picoshell Particles

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Production of high-energy fats by microalgae may provide a sustainable, renewable energy source that can help tackle climate change. However, microalgae engineered to produce lipids rapidly usually grow slowly themselves, making it difficult to increase overall yields. 

Production of high-energy fats by microalgae may provide a sustainable, renewable energy source that can help tackle climate change. However, microalgae engineered to produce lipids rapidly usually grow slowly themselves, making it difficult to increase overall yields. 

UCLA bioengineers have created a new type of petri dish in the form of microscopic, permeable particles that can dramatically speed up research and development (R&D) timelines of biological products, such as fatty acids for biofuels. Dubbed PicoShells, the picoliter (trillionth of a liter), porous, hydrogel particles can enable more than one million individual cells to be compartmentalized, cultured in production-relevant environments, and selected based on growth and biomass accumulation traits using standard cell-processing equipment. 

Proceedings of the National Academy of Sciences recently published a study detailing how PicoShells work and their potential applications.

PicoShells consist of a hollow inner cavity where cells are encapsulated and a porous outer shell that allows for continuous solution exchange with the external environment so that nutrients, cell-communication molecules and cytotoxic cellular byproducts can transport freely in and out of the inner cavity. The shell also keeps the small groups of growing cells penned in, allowing researchers to study and compare their behaviors — what they do, how fast they grow, what they produce — to those of other groups inside various PicoShells. 

This new class of lab tool allows researchers to grow living, single-cell microorganisms — including algae, fungi and bacteria — under the same industrial-production conditions, such as in a bioreactor filled with wastewater or an outdoor cultivation pond. 

“PicoShells are like very tiny mesh balloons. The growing cells inside them are effectively fenced in but not sealed off,” said study leader Dino Di Carlo, UCLA’s Armond and Elena Hairapetian Professor in Engineering and Medicine at the UCLA Samueli School of Engineering. “With this new tool, we can now study the individual behaviors of millions of living cells in the relevant environment. This could shorten R&D-to-commercial production timelines for bioproducts from a few years to a few months. PicoShells could also be a valuable tool for fundamental biology studies.” 

PicoShells’ permeability can bring the lab to the industrial environment, allowing testing at a sectioned-off area of a working facility. Growth can occur more quickly and cell strains that perform well can be identified and selected for further screening. 

According to the researchers, another advantage of this new tool is that the analysis of millions of PicoShells is automated since they are also compatible with standard lab equipment used for high-volume cell processing.

Massive groups of cells, up to 10 million in one day, can be sorted and organized by certain characteristics. Continuous analysis could result in ideal sets of cells — ones that are already performing well in the environment with suitable temperature, nutrient composition and other properties that could be used in mass production — in just a few days rather than the several months it would take using current technologies.

The shells can be engineered to burst when the cells inside have divided and grown beyond their peak volume. Those free cells are still viable and can be recaptured for continued research or further selection. The researchers can also create shells with chemical groups that break down when exposed to biocompatible reagent, enabling a multifaceted approach to release selected cells.

“If we want to zero in on algae that are the best at producing biofuels, we can use PicoShells to organize, grow and process millions of single algal cells,” said lead author Mark van Zee, a bioengineering graduate student at UCLA Samueli. “And we can do that in machines that sort them using fluorescent tags that light up to signify fuel levels.”

Currently, cultivating and comparing such microorganisms are done mostly using traditional lab tools, such as microwell plates — cartons that hold several dozen small test tube-like volumes. However, these methods are slow and it is difficult to quantify their effectiveness because it can take weeks or months to grow large colonies for study. Other approaches, such as water-in-oil droplet emulsions, can be used to analyze cells in smaller volumes, but surrounding oils prevent the free exchange of medium into the water drops. Even cells or microorganisms that perform well in lab conditions may not do as well once they are placed in industrial environments, such as bioreactors or outdoor cultivation farms. As a result, cell strains that are developed in the lab often do not exhibit the same beneficial characteristic behavior when transferred to industrial production. 

Microwell plates also are limited in the number of experiments that can be performed, resulting in a great deal of trial and error in finding cell strains that work sufficiently well for mass production.

The researchers demonstrated the new tool by growing colonies of algae and yeast, comparing their growth and viability against other colonies grown in water-in-oil emulsions. For the algae, the team found that PicoShell colonies accumulated biomass rapidly while algae did not grow at all in water-in-oil emulsions. Similar results were found in their yeast experiments. By selecting the top growing algae in PicoShells, the researchers could increase the production of chlorophyll biomass by 8% after only a single cycle. 

The authors said PicoShells could offer a faster alternative to develop new algae and yeast strains, leading to improved biofuels, plastics, carbon-capture materials and even food products and alcoholic beverages. Further refinements to the technology, such as coating the shells with antibodies, could also lead to developing new types of protein-based medicines.

Di Carlo, van Zee and study co-author Joseph de Rutte Ph.D. ’20, a former member of Di Carlo’s research group, are named inventors on a patent application filed by the UCLA Technology Development Group.

The other UCLA authors on the paper are Rose Rumyan, Cayden Williamson, Trevor Burnes, Andrew Sonico Eugenio, Sara Badih, Dong-Hyun Lee and Maani Archang. Randor Radakovits from Synthetic Genomics of San Diego is also an author.

The study was supported by the Presidential Early Career Award for Scientists and Engineers and a planning award from the California NanoSystems Institute (CNSI) at UCLA.

Di Carlo holds faculty appointments in bioengineering, and mechanical and aerospace engineering at UCLA Samueli. He is a member of CNSI and the Jonsson Comprehensive Cancer Center at UCLA.

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