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Electricity-Conducting Bacteria May Inspire Next-Gen Medical Devices

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Nanowires
Credit: Edward H. Egelman

Technological advances with potential for improving human health sometimes come from the most unexpected places. An intriguing example is an electricity-conducting biological nanowire that holds promise for powering miniaturized pacemakers and other implantable electronic devices.

The nanowires come from a bacterium called Geobacter sulfurreducens, shown in the electron micrograph above. This rod-shaped microbe (white) was discovered two decades ago in soil collected from an unlikely place: a ditch outside of Norman, Oklahoma. The bug can conduct electricity along its arm-like appendages, and, in the hydrocarbon-contaminated, oxygen-depleted soil in which it lives, such electrical inputs and outputs are essentially the equivalent of breathing.

Scientists fascinated with G. sulfurreducens thought that its electricity had to be flowing through well-studied microbial appendages called pili. But, as the atomic structure of these nanowires (multi-colors, foreground) now reveals, these nanowires aren’t pili at all! Instead, the bacteria have manufactured unique submicroscopic arm-like structures. These arms consist of long, repetitive chains of a unique protein, each surrounding a core of iron-containing molecules.

The surprising discovery, published in the journal Cell, was made by an NIH-funded team involving Edward Egelman, University of Virginia Health System, Charlottesville. Egelman’s lab has had a long interest in what’s called a type 4 pili. These strong, adhering appendages help certain infectious bacteria enter tissues and make people sick. In fact, they enable bugs like Neisseria meningitidis to cross the blood-brain barrier and cause potentially deadly bacterial meningitis. While other researchers had proposed that those same type 4 pili allowed G. sulfurreducens to conduct electricity, Egelman wasn’t so sure.

So, he took advantage of recent advances in cryo-electron microscopy, which involves flash-freezing molecules at extremely low temperatures before bombarding them with electrons to capture their images with a special camera. The cryo-EM images allowed his team to nail down the atomic structure of the nanowires, now called OmcS filaments.

Using those images and sophisticated bioinformatics, Egelman and team determined that OmcS proteins uniquely fit into the nanowires’ long repetitive chains, spacing their iron-bearing cores at regular intervals to transfer electrons and convey electricity. In fact, bacteria unable to produce OmcS proteins make filaments that conduct electricity 100 times less efficiently.

With these cryo-EM structures in hand, Egelman says his team will continue to explore their conductive properties. Such knowledge might someday be used to build biologically-inspired nanowires, measuring 1/100,000th the width of a human hair, to connect miniature electronic devices directly to living tissues. This is one more example of how nature’s ability to invent is pretty breathtaking—surely one wouldn’t have predicted the discovery of nanowires in a bacterium that lives in contaminated ditches.

Reference:

[1] Structure of Microbial Nanowires Reveals Stacked Hemes that Transport Electrons over Micrometers. Wang F, Gu Y, O’Brien JP, Yi SM, Yalcin SE, Srikanth V, Shen C, Vu D, Ing NL, Hochbaum AI, Egelman EH, Malvankar NS. Cell. 2019 Apr 4;177(2):361-369.

Links:

Electroactive microorganisms in bioelectrochemical systems. Logan BE, Rossi R, Ragab A, Saikaly PE. Nat Rev Microbiol. 2019 May;17(5):307-319.

High Resolution Electron Microscopy (National Cancer Institute/NIH)

Egelman Lab (University of Virginia, Charlottesville)

NIH Support: National Institute of General Medical Sciences; National Institute of Allergy and Infectious Diseases; Common Fund


Fighting Cancer with Next-Gen Cell Engineering

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Kole Roybal
Credit: Susan Merrell

Researchers continue to make progress with cancer immunotherapy, a type of treatment that harnesses the body’s own immune cells to attack cancer. But Kole Roybal wants to help move the field further ahead by engineering patients’ immune cells to detect an even broader range of cancers and then launch customized attacks against them.

With an eye toward developing the next generation of cell-based immunotherapies, this synthetic biologist at University of California, San Francisco, has already innovatively hacked into how certain cells communicate with each other. Now, he and his research team are using a 2018 NIH Director’s New Innovator Award to build upon that progress.

Roybal’s initial inspiration is CAR-T therapy, one of the most advanced immunotherapies to date. In CAR-T therapy, some of a cancer patient’s key immune cells, called T cells, are removed and engineered in a way that they begin to produce new surface proteins called chimeric antigen receptors (CARs). Those receptors allow the cells to recognize and attack cancer cells more effectively. After expanding the number of these engineered T cells in the lab, doctors infuse them back into patients to enhance their immune systems’s ability to seek-and-destroy their cancer.

As helpful as this approach has been for some people with leukemia, lymphoma, and certain other cancers, it has its limitations. For one, CAR-T therapy relies solely on a T cell’s natural activation program, which can be toxic to patients if the immune cells damage healthy tissues. In other patients, the response simply isn’t strong enough to eradicate a cancer.

Roybal realized that redirecting T cells to attack a broader range of cancers would take more than simply engineering the receptors to bind to cancer cells. It also would require sculpting novel immune cell responses once those receptors were triggered.

Roybal found a solution in a new class of lab-made receptors known as Synthetic Notch, or SynNotch, that he and his colleagues have been developing over the last several years [1, 2]. Notch protein receptors play an essential role in developmental pathways and cell-to-cell communication across a wide range of animal species. What Roybal and his colleagues found especially intriguing is the protein receptors’ mode of action is remarkably direct.

When a protein binds the Notch receptor, a portion of the receptor breaks off and heads for the cell nucleus, where it acts as a switch to turn on other genes. They realized that engineering a cancer patient’s immune cells with synthetic SynNotch receptors could offer extraordinary flexibility in customized sensing and response behaviors. What’s more, the receptors could be tailored to respond to a number of user-specified cues outside of a cell.

In his NIH-supported work, Roybal will devise various versions of SynNotch-engineered cells targeting solid tumors that have proven difficult to treat with current cell therapies. He reports that they are currently developing the tools to engineer cells to sense a broad spectrum of cancers, including melanoma, glioblastoma, and pancreatic cancer.

They’re also engineering cells equipped to respond to a tumor by producing a range of immune factors, including antibodies known to unleash the immune system against cancer. He says he’ll also work on adding engineered SynNotch molecules to other immune cell types, not just T cells.

Given the versatility of the approach, Roybal doesn’t plan to stop there. He’s also interested in regenerative medicine and in engineering therapeutic cells to treat autoimmune conditions. I’m looking forward to see just how far these and other next-gen cell therapies will take us.

References:

[1] Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Morsut L, Roybal KT, Xiong X, Gordley RM, Coyle SM, Thomson M, Lim WA. Cell. 2016 Feb 11;164(4):780-91.

[2] Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Roybal KT, Williams JZ, Morsut L, Rupp LJ, Kolinko I, Choe JH, Walker WJ, McNally KA, Lim WA. Cell. 2016 Oct 6;167(2):419-432.e16.

Links:

Car-T Cells: Engineering Patients’ Immune Cells to Treat Cancers (National Cancer Institute/NIH)

Synthetic Biology for Technology Development (National Institute of Biomedical Imaging and Bioengineering/NIH)

Roybal Lab (University of California, San Francisco)

Roybal Project Information (NIH RePORTER)

NIH Support: Common Fund; National Cancer Institute


Progress Toward 3D Printed Human Organs

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There’s considerable excitement that 3D printing technology might one day allow scientists to produce fully functional replacement organs from one’s own cells. While there’s still a lot to learn, this video shows just some of the amazing progress that’s now being made.

The video comes from a bioengineering team at Rice University, Houston, that has learned to bioprint the small air sacs in the lungs. When hooked up to a machine that pulsed air in and out of the air sacs, the rhythmic movement helped to mix red blood cells traveling through an associated blood vessel network. Those red cells also took up oxygen in much the way that blood vessels do when surrounding the hundreds of millions of air sacs in our lungs.

As mentioned in the video, one of the biggest technical hurdles in growing fully functional replacement tissues and organs is to find a way to feed the growing tissues with a blood supply and to remove waste products. In this study recently published in Science [1], the NIH-supported team cleared this hurdle by creating an open-source bioprinting technology they call SLATE, which is short for “stereo-lithography apparatus for tissue engineering.”

The SLATE system “grows” soft hydrogel scaffolds one layer at a time. Each layer is printed using a liquid pre-hydrogel solution that solidifies when exposed to blue light. By also projecting light into the hydrogel as a pixelated 3D shape, it’s possible to print complex 3D structures within minutes.

When the researchers first started, their printouts lacked the high resolution, submillimeter-scale channels needed to generate intricate vascular networks. In other manufacturing arenas, light-absorbing chemicals have helped control the conversion from liquid to solid in a very fine polymer layer. But these industrial light-absorbing chemicals are highly toxic and therefore unsuitable for scaffolds that grow living tissues and organs.

The researchers, including Bagrat Grigoryan, Jordan Miller, and Kelly Stevens, wondered whether they could swap out those noxious ingredients with synthetic and natural food dyes widely used in the food industry. These dyes include curcumin, anthocyanin, and tartrazine (yellow dye #5). Their studies showed that those fully biocompatible dyes worked as effective light absorbers, allowing the scientists to recreate the complex architectures of human vasculature. Importantly, the living cells survived within the soft scaffold!

These models are already yielding intriguing new insights into the vascular structures found within our organs and how those architectures may influence function in ways that hadn’t been well understood. In the near term, tissues and organs grown on such scaffolds might also find use as sophisticated, 3D tissue “chips,” with potential for use in studies to predict whether drugs will be safe in humans.

In the long term, this technology may allow production of replacement organs from those needing them. More than 100,000 men, women, and children are on the national transplant waiting list in the United States alone and 20 people die each day waiting for a transplant [2]. Ultimately, with the aid of bioprinting advances like this one, perhaps one day we’ll have a ready supply of perfectly matched and fully functional organs.

References:

[1] Multivascular networks and functional intravascular topologies within biocompatible hydrogels. Grigoryan B, Paulsen SJ, Corbett DC, Sazer DW, Fortin CL, Zaita AJ, Greenfield PT, Calafat NJ, Gounley JP, Ta AH, Johansson F, Randles A, Rosenkrantz JE, Louis-Rosenberg JD, Galie PA, Stevens KR, Miller JS. Science. 2019 May 3;364(6439):458-464.

[2] Organ Donor Statistics, Health Resources & Services Administration, October 2018.

Links:

Tissue Engineering and Regenerative Medicine (National Institute of Biomedical Imaging and Bioengineering/NIH)

Tissue Chip for Drug Screening (National Center for Advancing Translational Sciences/NIH)

Miller Lab (Rice University, Houston)

NIH Support: National Heart, Lung, and Blood Institute; National Institute of Biomedical Imaging and Bioengineering; National Institute of General Medical Sciences; Common Fund


Combating Mosquitoes with an Engineered Fungus

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Caption: Anopheles coluzzii mosquito with transgenic fungus (green) emerging from its body after death. Credit: Brian Lovett, University of Maryland, College Park

Almost everywhere humans live on this planet, mosquitoes carry microbes that cause potentially deadly diseases, from West Nile virus to malaria. While chemical insecticides offer a line of defense, mosquito populations often grow resistant to them. So, it’s intriguing to learn that we may now have another ally in this important fight: a genetically engineered fungus!

Reporting in the journal Science, an international research team supported by NIH describes how this new approach might be used to combat malaria [1]. A fungus called Metarhizium pingshaense is a natural enemy of the mosquito, but, by itself, it kills mosquitoes too slowly to control transmission of malaria. To make this fungus an even more efficient mosquito killer, researchers engineered it to carry a gene encoding a toxin, derived from a spider, that is deadly to insects. Tests of the souped-up fungus in a unique contained facility designed to simulate a West African village found it safely and rapidly killed insecticide-resistant mosquitoes, reducing their numbers by more than 99 percent within 45 days.

Mosquitoes are the deadliest animals in the world. More than 3.2 billion people—about half of all humans—are at risk for malaria, and more than 400,000 die each year from the disease. Other mosquito-borne illnesses, including Zika and dengue viruses, sicken millions more each year. By combining existing insect control strategies with the latest technical innovation, it should be possible to lower those numbers.

In the latest study, Raymond St. Leger and Brian Lovett, University of Maryland, College Park, teamed with Abdoulaye Diabate and colleagues from Institut de Recherche en Sciences de la Santé/Cente Muraz, Burkina Faso, West Africa. The researchers employed a strategy that’s been in use around the world for more than 100 years to control agricultural pests.

The approach involves the fungal species Metarhizium, which kills a variety of insects. Earlier studies had shown that spores from a specific Metarhizium strain could make a big enough dent in a mosquito population to raise the possibility of using the fungus to reduce infective bites among humans [2]. But killing off the mosquitoes required very large quantities of fungal spores and usually took a couple of weeks.

Here’s where things turned innovative. To boost the fungus’s potency, St. Leger and colleagues used genetic engineering to add a toxin derived from the Australian Blue Mountains funnel-web spider. The toxin came with a major advantage: the U.S. Environmental Protection Agency (EPA) already has approved its use as a safe-and-effective insecticidal protein.

Besides giving the engineered fungus that ability to produce a spider toxin, the researchers added another clever element. They didn’t want the fungus to produce the toxin all the time—only after it comes in contact with a mosquito’s hemolymph, the insect equivalent of blood. So, they needed to insert a control switch, and the researchers knew just where to find the needed part.

Once inside a mosquito, the fungus naturally produces a structural protein called collagen that shields it from the insect’s immune system. A genetic switch that turns “on” when it detects an insect’s hemolymph controls that collagen production. To ensure that the spider toxin was produced at just the right time, the researchers hotwired their Metarhizium to begin producing it under the control of this same genetic switch.

The next step was to test this modified organism in a more natural, but controlled, environment. The researchers spent more than a year in Burkina Faso building a specialized facility called a MosquitoSphere. It’s similar to a very large greenhouse, but with mosquito netting instead of glass.

The MosquitoSphere has six separate compartments, four of which contain West African huts, along with native plants and breeding sites for mosquitoes. The researchers hung a black cotton sheet, previously soaked in sesame oil, on the wall of a hut in each of three chambers.

In one hut, the sesame oil contained genetically engineered fungal spores. In the second hut, the oil contained natural fungal spores. In the third hut, there were no spores at all. Then, they released 1,000 adult male and 500 adult female mosquitoes into each chamber and watched what happened over the next 45 days.

In the hut without spores, the mosquitoes established a stable population of almost 1,400. In the chamber with the natural spores, 450 mosquitoes survived. But, in the chamber with the engineered fungus, the researchers counted just 13 survivors—too few to sustain a viable population.

The researchers say they suspect the fungus would be relatively easy to contain in nature. It’s sticky and not easily airborne. The spores are also extremely sensitive to sunlight, making it difficult for them to travel far. Importantly, the fungus didn’t harm other beneficial insects, including honeybees.

Caution is warranted before considering the release of a genetically engineered organism into the wild. In the meantime, the genetically engineered fungus also will serve as a platform for continued technology development.

The system can be readily adapted to target mosquitoes or other insects , perhaps using different natural toxins if insects might grow resistant to Metarhizium just as they have to traditional insecticides. Interestingly, the researchers note that the engineered fungi appear to make mosquitoes sensitive to chemical insecticides again, suggesting that the two types of insect-killers might be used successfully in combination.

References:

[1] Transgenic Metarhizium rapidly kills mosquitoes in a malaria-endemic region of Burkina Faso. Lovett B, Bilgo E, Millogo SA, Ouattarra AK, Sare I, Gnambani EJ, Dabire RK, Diabate A, St Leger RJ. Science. 2019 May 31;364(6443):894-897.

[2] An entomopathogenic fungus for control of adult African malaria mosquitoes. Scholte EJ, Ng’habi K, Kihonda J, Takken W, Paaijmans K, Abdulla S, Killeen GF, Knols BG. Science. 2005 Jun 10;308(5728):1641-2.

Links:

Transgenic Fungus Rapidly Killed Malaria Mosquitoes in West African Study (University of Maryland News Release)

Malaria (National Institute of Allergy and Infectious Diseases/NIH)

Funnel-Web Spiders (Australian Museum, Sydney)

Video: 2016 Grand Challenges Spotlight Talk: Abdoulaye Diabaté (YouTube)

Raymond St. Leger (University of Maryland, College Park)

NIH Support: National Institute of Allergy and Infectious Diseases


An ‘Off-the-Shelf’ Replacement for Damaged Blood Vessels?

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human acellular vessel
Credit: Humacyte, Inc., Durham, NC

The object in the image above might look like an ordinary plastic tube. But this tube is neither plastic nor ordinary. It’s a bioengineered replacement human blood vessel that could one day benefit people who receive kidney dialysis or undergo coronary bypass surgery.

It’s called a human acellular vessel (HAV), and an NIH-funded team, led by Heather Prichard, Humacyte Inc., Durham, NC, grows these acellular vessels. They can run up to about 16-inches long with a diameter of 0.2 inches, which is well within the range of a human blood vessel.

Prichard and team start with a lightweight and biodegradable polymer mesh. They then seed the mesh scaffold with cells taken from human donor tissue within a 3D bioreactor system in the lab. The system is specially designed to provide nutrients and mechanical pulsations similar to those present in an intact human circulatory system.

After incubating the growing vessels for eight weeks, the researchers remove all the living cells, leaving behind mostly human collagen, a fibrous protein and major structural component of a blood vessel wall. It forms a non-living, replacement vessel that retains the physical and mechanical integrity of a human blood vessel. But, because these HAVs don’t have cells, they potentially can be surgically implanted into any human patient without risk of an immune reaction.

As reported recently in Science Translational Medicine, the best part is what happens after an HAV is implanted into the body [1]. The patient’s own cells infiltrate the HAVs. Over the course of many weeks, these cells produce multiple layers of living tissue to transform the acellular HAV into a functional, living blood vessel.

So far, HAVs have been tested in more than 240 people with end-stage kidney failure. The HAVs were implanted into the upper arms of participants and remained there from 16 to 200 weeks while these patients underwent dialysis three times per week to filter waste products from their blood. The early results indicate these bioengineered blood vessels were safe and fully functional. More research, though, will be needed to ensure that’s indeed the case.

For people who receive kidney dialysis, doctors now typically access the vasculature by linking an artery to a vein under the skin of the arm, making an “AV fistula.” But doctors can also use the HAV tube to make the needed connection.

What’s potentially game changing about HAVs is they offer the same “off-the-shelf” ease of a plastic tube but with the advantages of living tissue. Those advantages include the ability to fight infection and self-heal from the inevitable injury that comes with repeated needle pokes.

Though most of the work to date has focused on people undergoing kidney dialysis, an ongoing clinical trial is testing the potential of HAVs to improve blood flow when surgically implanted into the legs of patients with peripheral arterial disease [3]. Prichard also sees potential for HAVs in heart surgery. For example, HAVs might be useful during coronary bypass surgery to repair a narrowed or blocked blood vessel. They could also be used to replace blood vessels damaged or missing due to congenital defects or traumatic injuries. Not bad for an object that looks like an ordinary plastic tube.

References:

[1] Bioengineered human acellular vessels recellularize and evolve into living blood vessels after human implantation. Kirkton RD, Santiago-Maysonet M, Lawson JH, Tente WE, Dahl SLM, Niklason LE, Prichard HL. Sci Transl Med. 2019 Mar 27;11(485).

[2] Kidney Disease Statistics for the United States. National Institute of Diabetes and Digestive and Kidney Diseases/NIH

[3] Humacyte’s HAV for Femoro-Popliteal Bypass in Patients With PAD. Clinicaltrials.gov

Links:

Safety and Efficacy of a Vascular Prosthesis for Hemodialysis Access in Patients With End-Stage Renal Disease (ClinicalTrials.Gov)

Hemodialysis (National Institute of Diabetes and Digestive and Kidney Diseases/NIH)

Tissue Engineering and Regenerative Medicine (National Institute of Biomedical Imaging and Bioengineering/NIH)

Humacyte (Durham, NC)

NIH Support: National Heart, Lung, and Blood Institute


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