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Creative Minds

Tackling Fibrosis with Synthetic Materials

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April Kloxin
April Kloxin/Credit: Evan Krape, University of Delaware, Newark

When injury strikes a limb or an organ, our bodies usually heal quickly and correctly. But for some people, the healing process doesn’t shut down properly, leading to excess fibrous tissue, scarring, and potentially life-threatening organ damage.

This permanent scarring, known as fibrosis, can occur in almost every tissue of the body, including the heart and lungs. With support from a 2019 NIH Director’s New Innovator Award, April Kloxin is applying her expertise in materials science and bioengineering to build sophisticated fibrosis-in-a-dish models for unraveling this complex process in her lab at the University of Delaware, Newark.

Though Kloxin is interested in all forms of fibrosis, she’s focusing first on the incurable and often-fatal lung condition called idiopathic pulmonary fibrosis (IPF). This condition, characterized by largely unexplained thickening and stiffening of lung tissue, is diagnosed in about 50,000 people each year in the United States.

IPF remains poorly understood, in part because it often is diagnosed when the disease is already well advanced. Kloxin hopes to turn back the clock and start to understand the disease at an earlier stage, when interventions might be more successful. The key is to develop a model that better recapitulates the complexity and irreversibility of the disease process in people.

Building that better model starts with simulating the meshwork of collagen and other proteins in the extracellular matrix (ECM) that undergird every tissue and organ in the body. The ECM’s interactions with our cells are essential in wound healing and, when things go wrong, also in causing fibrosis.

Kloxin will build three-dimensional hydrogels, crosslinked sponge-like networks of polymers, peptides, and proteins, with structures that more accurately capture the biological complexities of human tissues, including the ECMs within fibrous collagen-rich microenvironments. Her synthetic matrices can be triggered with light to lock in place and stiffen. The matrices also will make it possible to culture the lung’s epithelium, or outermost layer of cells, and connective tissue that surrounds it, to study cellular responses as the model shifts from a healthy and flexible to a stiffened, disease-like state.

Kloxin and her team will also integrate into their model system lung cells that have been engineered to fluoresce or light up under a microscope when the wound-healing program activates. Such fluorescent reporters will allow her team to watch for the first time how different cells and their nearby microenvironment respond as the composition of the ECM changes and stiffens. With this system, she’ll also be able to search for small molecules with the ability to turn off excessive wound healing.

The hope is that what’s learned with her New Innovator Award will lead to fresh insights and ultimately new treatments for this mysterious, hard-to-treat condition. But the benefits could be even more wide-ranging. Kloxin thinks that her findings will have implications for the prevention and treatment of other fibrotic diseases as well.

Links:

Idiopathic Pulmonary Fibrosis (National Heart, Lung, and Blood Institute/NIH)

April Kloxin Group (University of Delaware, Newark)

Kloxin Project Information (NIH RePORTER)

NIH Director’s New Innovator Award (Common Fund)

NIH Support: Common Fund; National Heart, Lung, and Blood Institute


Could A Gut-Brain Connection Help Explain Autism?

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What is Your Big Idea?
Diego Bohórquez/Credit: Duke University, Durham, NC

You might think nutrient-sensing cells in the human gastrointestinal (GI) tract would have no connection whatsoever to autism spectrum disorder (ASD). But if Diego Bohórquez’s “big idea” is correct, these GI cells, called neuropods, could one day help to provide a direct link into understanding and treating some aspects of autism and other brain disorders.

Bohórquez, a researcher at Duke University, Durham, NC, recently discovered that cells in the intestine, previously known for their hormone-releasing ability, form extensions similar to neurons. He also found that those extensions connect to nerve fibers in the gut, which relay signals to the vagus nerve and onward to the brain. In fact, he found that those signals reach the brain in milliseconds [1].

Bohórquez has dedicated his lab to studying this direct, high-speed hookup between gut and brain and its impact on nutrient sensing, eating, and other essential behaviors. Now, with support from a 2019 NIH Director’s New Innovator Award, he will also explore the potential for treating autism and other brain disorders with drugs that act on the gut.

Bohórquez became interested in autism and its possible link to the gut-brain connection after a chance encounter with Geraldine Dawson, director of the Duke Center for Autism and Brain Development. Dawson mentioned that autism typically affects multiple organ systems.

With further reading, he discovered that kids with autism frequently cope with GI issues, including bowel inflammation, abdominal pain, constipation, and/or diarrhea [2]. They often also show unusual food-related behaviors, such as being extremely picky eaters. But his curiosity was especially piqued by evidence that certain gut microbes can influence abnormal behaviors in mice that model autism.

With his New Innovator Award, Bohórquez will study neuropods and the gut-brain connection in a mouse model of autism. Using the tools of optogenetics, which make it possible to activate cells with light, he’ll also see whether autism-like symptoms in mice can be altered or alleviated by controlling neuropods in the gut. Those symptoms include anxiety, repetitive behaviors, and lack of interest in interacting with other mice. He’ll also explore changes in the animals’ eating habits.

In another line of study, he will take advantage of intestinal tissue samples collected from people with autism. He’ll use those tissues to grow and then examine miniature intestinal “organoids,” looking for possible evidence that those from people with autism are different from others.

For the millions of people now living with autism, no truly effective drug therapies are available to help to manage the condition and its many behavioral and bodily symptoms. Bohórquez hopes one day to change that with drugs that act safely on the gut. In the meantime, he and his fellow “GASTRONAUTS” look forward to making some important and fascinating discoveries in the relatively uncharted territory where the gut meets the brain.

References:

[1] A gut-brain neural circuit for nutrient sensory transduction. Kaelberer MM, Buchanan KL, Klein ME, Barth BB, Montoya MM, Shen X, Bohórquez DV. Science. 2018 Sep 21;361(6408).

[2] Association of maternal report of infant and toddler gastrointestinal symptoms with autism: evidence from a prospective birth cohort. Bresnahan M, Hornig M, Schultz AF, Gunnes N, Hirtz D, Lie KK, Magnus P, Reichborn-Kjennerud T, Roth C, Schjølberg S, Stoltenberg C, Surén P, Susser E, Lipkin WI. JAMA Psychiatry. 2015 May;72(5):466-474.

Links:

Autism Spectrum Disorder (National Institute of Mental Health/NIH)

Bohórquez Lab (Duke University, Durham, NC)

Bohórquez Project Information (NIH RePORTER)

NIH Director’s New Innovator Award (Common Fund)

NIH Support: Common Fund; National Institute of Mental Health


Finding New Genetic Mutations Amid Healthy Cells

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Po-Ruh Loh
Po-Ru Loh Courtesy of Loh Lab

You might recall learning in biology class that the cells constantly replicating and dividing in our bodies all carry the same DNA, inherited in equal parts from each parent. But it’s become increasingly clear in recent years that even seemingly healthy tissues contain neighborhoods of cells bearing their own acquired genetic mutations. The question is: What do all those altered cells mean for our health?

With support from a 2018 NIH Director’s New Innovator Award, Po-Ru Loh, Harvard Medical School, Boston, is on a quest to find out, though without the need for sequencing lots of DNA in his own lab. Loh will instead develop ultrasensitive computational tools to pick up on those often-subtle alterations within the vast troves of genomic data already stored in databases around the world.

How is that possible? The math behind it might be complex, but the underlying idea is surprisingly simple. His algorithms look for spots in the genome where a slight imbalance exists in the quantity of DNA inherited from mom versus dad.

Actually, Loh can’t tell from the data which parent provided any snippet of chromosomal DNA. But looking at DNA sequenced from a mixture of many cells, he can infer which stretches of DNA were most likely inherited together from a single parent.

Any slight skew in those quantities point the way to genomic territory where a tiny portion of chromosomal DNA either went missing or became duplicated in some cells. This common occurrence, especially in older adults, leads to a condition called genetic mosaicism, meaning that, contrary to most biology textbooks, all cells aren’t exactly the same.

By detecting those subtle imbalances in the data, Loh can pinpoint small DNA alterations, even when they occur in 1 in 1,000 cells collected from a person’s bloodstream, saliva, or tissues. That’s the kind of sensitivity that most scientists would not have thought possible.

Loh has already begun putting his new computational approach to work, as reported in Nature last year [1]. In DNA data from blood samples of more than 150,000 participants in the United Kingdom Biobank, his method uncovered well over 8,000 mosaic chromosomal alterations.

The study showed that some of those alterations were associated with an increased risk of developing blood cancers. However, it’s important to note that most people with evidence of mosaicism won’t go on to develop cancer. The researchers also made the unexpected discovery that some individuals carried genetic variants that made them more prone than others to pick up new mutations in their blood cells.

What’s especially exciting is Loh’s computational tools now make it possible to search for signs of mosaicism within all the genetic data that’s ever been generated. Even more importantly, these tools will allow Loh and other researchers to ask and answer important questions about the consequences of mosaicism for a wide range of diseases.

Reference:

[1] Insights into clonal haematopoiesis from 8,342 mosaic chromosomal alterations. Loh PR, Genovese G, Handsaker RE, Finucane HK, Reshef YA, Palamara PF, Birmann BM, Talkowski ME, Bakhoum SF, McCarroll SA, Price AL. Nature. 2018 Jul;559(7714):350-355.

Links:

Loh Lab (Harvard Medical School, Boston)

Loh Project Information (NIH RePORTER)

NIH Director’s New Innovator Award (Common Fund)

NIH Support: Common Fund; National Institute of Environmental Health Sciences


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


Deciphering Another Secret of Life

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Srivatsan Raman
Credit: Robin Davies, University of Wisconsin-Madison

In 1953, Francis Crick famously told the surprised customers at the Eagle and Child pub in London that he and Jim Watson had discovered the secret of life. When NIH’s Marshall Nirenberg and his colleagues cracked the genetic code in 1961, it was called the solution to life’s greatest secret. Similarly, when the complete human genome sequence was revealed for the first time in 2003, commentators (including me) referred to this as the moment where the book of life for humans was revealed. But there are many more secrets of life that still need to be unlocked, including figuring out the biochemical rules of a protein shape-shifting phenomenon called allostery [1].

Among those taking on this ambitious challenge is a recipient of a 2018 NIH Director’s New Innovator Award, Srivatsan Raman of the University of Wisconsin-Madison. If successful, such efforts could revolutionize biology by helping us better understand how allosteric proteins reconfigure themselves in the right shapes at the right times to regulate cell signaling, metabolism, and many other important biological processes.

What exactly is an allosteric protein? Proteins have active, or orthosteric, sites that turn the proteins off or on when specific molecules bind to them. Some proteins also have less obvious regulatory, or allosteric, sites that indirectly affect the proteins’ activity when outside molecules bind to them. In many instances, allosteric binding triggers a change in the shape of the protein.

Allosteric proteins include oxygen-carrying hemoglobin and a variety of enzymes crucial to human health and development. In his work, Raman will start by studying a relatively simple bacterial protein, consisting of less than 200 amino acids, to understand the basics of how allostery works over time and space.

Raman, who is a synthetic biologist, got the idea for this project a few years ago while tinkering in the lab to modify an allosteric protein to bind new molecules. As part of the process, he and his team used a new technology called deep mutational scanning to study the functional consequences of removing individual amino acids from the protein [2].

The screen took them on a wild ride of unexpected functional changes, and a new research opportunity called out to him. He could combine this scanning technology with artificial intelligence and other cutting-edge imaging and computational tools to probe allosteric proteins more systematically in hopes of deciphering the basic molecular rules of allostery.

With the New Innovator Award, Raman’s group will first create a vast number of protein mutants to learn how best to determine the allosteric signaling pathway(s) within a protein. They want to dissect out the properties of each amino acid and determine which connect into a binding site and precisely how those linkages are formed. The researchers also want to know how the amino acids tend to configure into an inactive state and how that structure changes into an active state.

Based on these initial studies, the researchers will take the next step and use their dataset to predict where allosteric pathways are found in individual proteins. They will also try to figure out if allosteric signals are sent in one direction only or whether they can be bidirectional.

The experiments will be challenging, but Raman is confident that they will serve to build a more unified view of how allostery works. In fact, he hopes the data generated—and there will be a massive amount—will reveal novel sites to control or exploit allosteric signaling. Such information will not only expand fundamental biological understanding, but will accelerate efforts to discover new therapies for diseases, such as cancer, in which disruption of allosteric proteins plays a crucial role.

References:

[1] Allostery: an illustrated definition for the ‘second secret of life.’ Fenton AW. Trends Biochem Sci. 2008 Sep;33(9):420-425.

[2] Engineering an allosteric transcription factor to respond to new ligands. Taylor ND, Garruss AS, Moretti R, Chan S, Arbing MA, Cascio D, Rogers JK, Isaacs FJ, Kosuri S, Baker D, Fields S, Church GM, Raman S. Nat Methods. 2016 Feb;13(2):177-183.

Links:

Drug hunters explore allostery’s advantages. Jarvis LM, Chemical & Engineering News. 2019 March 10

Allostery: An Overview of Its History, Concepts, Methods, and Applications. Liu J, Nussinov R. PLoS Comput Biol. 2016 Jun 2;12(6):e1004966.

Srivatsan Raman (University of Wisconsin-Madison)

Raman Project Information (NIH RePORTER)

NIH Director’s New Innovator Award (Common Fund/NIH)

NIH Support: National Institute of General Medical Sciences; Common Fund


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