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New synthetic molecular prosthetic cell acts as AND gate for disease treatment

“Cytokine converter” AND-gate synthetic-biology prosthesis used to treat psoriasis in mice. Top left: skin before; right: skin after. (credit: Lina Schukur et al./Science Translational Medicine)

An advanced “molecular prosthetic” — a cell with synthetic gene circuits that can be implanted into an organism to take over metabolic functions that the organism cannot perform itself — has been developed by  ETH Zurich scientists.

Previous gene circuits typically monitored only whether one disease-causing molecule (called a cytokine) was present in their environment and if so, produced a single therapeutic cytokine as a response. The new “cytokine converter” synthetic circuit functions like an AND gate: It can detect two different cytokines simultaneously, and if (and only if) both are present, produces two different cytokines that can treat a disease.

As a feasibility study, Professor Martin Fussenegger and his team at ETH’s Department of Biosystems Science and Engineering in Basel used the new cytokine converter prosthesis to treat psoriasis — a complex, chronic inflammatory disease of the skin with no cure — in mice.*

When the cytokine converter detected both of the inflammatory molecules TNF and IL-22, the synthetic gene circuit produced the anti-inflammatory cytokine molecules IL-4 and IL-10, suppressing the inflammatory response.

Preventive treatment for possible wide range of diseases

The symptoms of psoriasis — inflamed, itchy and sometimes flaky areas of skin — are usually combated with a locally applied ointment. “This means that with the existing therapies, we are practically always lagging behind the symptoms,” says Fussenegger.

The gene circuit implant, on the other hand, allowed for prevention: “The circuit begins producing anti-inflammatory messengers at an early stage — when a phase is looming at the level of inflammatory messengers, instead of waiting until skin rashes appear,” he said. It prevented psoriasis “flare-ups” but also treated acute (established) psoriasis, returning skin to normal in mice.

Fussenegger said such molecular prosthetics may one day be implanted in psoriasis patients. However, since growth in connective tissue could cut the implant off from the bloodstream over time, a doctor would probably have to replace it every few months.

Biological circuits with AND gates may also be suitable for other diseases, he said. “Chronic inflammatory diseases are a good example of the type of disease that cannot be diagnosed by measuring a single molecule. However, generally such diseases could be diagnosed using a designer cell that measures the profile of several messengers in the bloodstream. And if this designer cell were also to produce therapeutic molecules, it would open up promising treatment options for a wide range of diseases in the future.”

* Psoriasis is associated with an increased risk of immune-mediated diseases, such as Crohn’s disease and ulcerative colitis, as well as certain cancers (liver and pancreatic), metabolic disorders (obesity and diabetes), and cardiovascular diseases, the authors note.

Abstract of Implantable synthetic cytokine converter cells with AND-gate logic treat experimental psoriasis

Taking pills may go the way of the horse and buggy, the rotary phone, and the Walkman, at least if synthetic biology has anything to say about it. Schukur et al. designed a circuit that would automatically sense the presence of two disease-causing molecules, called cytokines, in the body and respond by triggering the production of two other cytokines that would treat the disease. This circuit was genetically engineered in a mammalian cell; in turn, the cell was implanted in mice with psoriasis—an inflammatory skin condition that has no cure. When levels of the proinflammatory cytokines TNF and IL22 peaked in the body, the synthetic circuit kicked into gear, converting these cytokine signals into an anti-inflammatory cellular output, consisting of IL4 and IL10, which then attenuated disease. The “cytokine converter” cells not only prevented psoriasis flare-ups, as they’re called, but also treated acute (established) psoriasis, returning skin to normal in mice. In demonstrating that the converter cells were responsive to blood from psoriasis patients, the authors suggest that synthetic biology may be ready to autonomously flip therapeutic switches in people and later take on other diseases with defined disease indicators.

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Hybrid solid-state chips and biological cells integrated at molecular level

Illustration depicting a biocell attached to a CMOS integrated circuit with a membrane containing sodium-potassium pumps in pores. Energy is stored chemically in ATP molecules. When the energy is released as charged ions (which are then converted to electrons to power the chip at the bottom of the experimental device), the ATP is converted to ADP + inorganic phosphate. (credit: Trevor Finney and Jared Roseman/Columbia Engineering)

Columbia Engineering researchers have combined biological and solid-state components for the first time, opening the door to creating entirely new artificial biosystems.

In this experiment, they used a biological cell to power a conventional solid-state complementary metal-oxide-semiconductor (CMOS) integrated circuit. An artificial lipid bilayer membrane containing adenosine triphosphate (ATP)-powered ion pumps (which provide energy for cells) was used as a source of ions (which were converted to electrons to power the chip).

The study, led by Ken Shepard, Lau Family Professor of Electrical Engineering and professor of biomedical engineering at Columbia Engineering, was published online today (Dec. 7, 2015) in an open-access paper in Nature Communications.

How to build a hybrid biochip

Living systems achieve this functionality with their own version of electronics based on lipid membranes and ion channels and pumps, which act as a kind of “biological transistor.” Charge in the form of ions carry energy and information, and ion channels control the flow of ions across cell membranes.

Solid-state systems, such as those in computers and communication devices, use electrons; their electronic signaling and power are controlled by field-effect transistors.

To build a prototype of their hybrid system, Shepard’s team packaged a CMOS integrated circuit (IC) with an ATP-harvesting “biocell.” In the presence of ATP, the system pumped ions across the membrane, producing an electrical potential (voltage)* that was harvested by the integrated circuit.

“We made a macroscale version of this system, at the scale of several millimeters, to see if it worked,” Shepard notes. “Our results provide new insight into a generalized circuit model, enabling us to determine the conditions to maximize the efficiency of harnessing chemical energy through the action of these ion pumps. We will now be looking at how to scale the system down.”

While other groups have harvested energy from living systems, Shepard and his team are exploring how to do this at the molecular level, isolating just the desired function and interfacing this with electronics. “We don’t need the whole cell,” he explains. “We just grab the component of the cell that’s doing what we want. For this project, we isolated the ATPases because they were the proteins that allowed us to extract energy from ATP.”

The capability of a bomb-sniffing dog, no Alpo required

Next, the researchers plan to go much further, such as recognizing specific molecules and giving chips the potential to taste and smell.

The ability to build a system that combines the power of solid-state electronics with the capabilities of biological components has great promise, they believe. “You need a bomb-sniffing dog now, but if you can take just the part of the dog that is useful — the molecules that are doing the sensing — we wouldn’t need the whole animal,” says Shepard.

The technology could also provide a power source for implanted electronic devices in ATP-rich environments such as inside living cells, the researchers suggest.

*  “In general, integrated circuits, even when operated at the point of minimum energy in subthreshold, consume on the order of 10−2 W mm−2 (or assuming a typical silicon chip thickness of 250 μm, 4 × 10−2 W mm−3). Typical cells, in contrast, consume on the order of 4 × 10−6 W mm−3. In the experiment, a typical active power dissipation for the IC circuit was 92.3 nW, and the active average harvesting power was 71.4 fW for the biocell (the discrepancy is managed through duty-cycled operation of the IC).” — Jared M. Roseman et al./Nature Communications

 

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Engineered bacteria form multicellular circuit to control protein expression

Two strains of synthetically engineered bacteria cooperate to create multicellular phenomena. Their fluorescence indicates the engineered capabilities have been activated. (credit: Bennett Lab/Rice University)

Rice University scientists and associates have created a biological equivalent to a computer circuit using multiple types of bacteria that change protein expression. The goal is to modify biological systems by controlling how bacteria influence each other. This could lead to bacteria that, for instance, beneficially alter the gut microbiome (collection of microorganisms) in humans.

The research is published in the journal Science.

Humans’ stomachs have a lot of different kinds of bacteria contained in the microbiome. “They naturally form a large consortium,” said Rice synthetic biologist Matthew Bennett. The idea is to engineer bacteria to be part of a consortium. “Working together allows them to effect more change than if they worked in isolation.”

In the proof-of-concept study, Bennett and his team created two strains of genetically engineered bacteria that regulate the production of proteins essential to intercellular signaling pathways, which allow cells to coordinate their efforts, generally in beneficial ways.

The synthetic microbial consortium oscillator yo-yo

The activator strain up-regulates genes in both strains; the repressor strain down-regulates genes in both strains, generating an oscillation of gene transcription in the bacterial population (credit: Ye Chen et al.)

“The main push in synthetic biology has been to engineer single cells,” Bennett said. “But now we’re moving toward multicellular systems. We want cells to coordinate their behaviors in order to elicit a populational response, just the way our bodies do.”

Bennett and his colleagues achieved their goal by engineering common Escherichia coli bacteria. By creating and mixing two genetically distinct populations, they prompted the bacteria to form a consortium.

The bacteria worked together by doing opposite tasks: One was an activator that up-regulated the expression of targeted genes; the other was a repressor that down-regulated specific genes. Together, they created oscillations of gene transcription in the bacterial population.

The two novel strains of bacteria sent out intercellular signaling molecules and created linked positive and negative feedback loops that affected gene production in the entire population. Both strains were engineered to make fluorescent reporter genes so their activities could be monitored. The bacteria were confined to microfluidic devices in the lab, where they could be monitored easily during each hours-long experiment.

When the bacteria were cultured in isolation, the protein oscillations did not appear, the researchers wrote.

Programmed yogurt, anyone?

Bennett said his lab’s work will help researchers understand how cells communicate, an important factor in fighting disease. “We have many different types of cells in our bodies, from skin cells to liver cells to pancreatic cells, and they all coordinate their behaviors to make us work properly,” he said. “To do this, they often send out small signaling molecules that are produced in one cell type and effect change in another cell type.

“We take that principle and engineer it into these very simple organisms to see if we can understand and build multicellular systems from the ground up.”

Ultimately, people might ingest the equivalent of biological computers that can be programmed through one’s diet, Bennett said. “One idea is to create a yogurt using engineered bacteria,” he said. “The patient eats it and the physician controls the bacteria through the patient’s diet. Certain combinations of molecules in your food can turn systems within the synthetic bacteria on and off, and then these systems can communicate with each other to effect change within your gut.”

KAIST and University of Houston scientists were also involved in the research. The National Institutes of Health, the Robert A. Welch Foundation, the Hamill Foundation, the National Science Foundation, and the China Scholarship Council supported the research.

Abstract of Emergent genetic oscillations in a synthetic microbial consortium

A challenge of synthetic biology is the creation of cooperative microbial systems that exhibit population-level behaviors. Such systems use cellular signaling mechanisms to regulate gene expression across multiple cell types. We describe the construction of a synthetic microbial consortium consisting of two distinct cell types—an “activator” strain and a “repressor” strain. These strains produced two orthogonal cell-signaling molecules that regulate gene expression within a synthetic circuit spanning both strains. The two strains generated emergent, population-level oscillations only when cultured together. Certain network topologies of the two-strain circuit were better at maintaining robust oscillations than others. The ability to program population-level dynamics through the genetic engineering of multiple cooperative strains points the way toward engineering complex synthetic tissues and organs with multiple cell types.