Study Describes a Biological Transistor for Computing Within Living Cells

Study Describes a Biological Transistor for Computing Within Living Cells

In a newly published study, Stanford researchers describe a biological transistor that is made from genetic material (DNA and RNA) in place of gears or electrons.

When Charles Babbage prototyped the first computing machine in the 19th century, he imagined using mechanical gears and latches to control information. ENIAC, the first modern computer developed in the 1940s, used vacuum tubes and electricity. Today, computers use transistors made from highly engineered semiconducting materials to carry out their logical operations.

And now a team of Stanford University bioengineers has taken computing beyond mechanics and electronics into the living realm of biology. In a paper published March 28 in Science, the team details a biological transistor made from genetic material — DNA and RNA — in place of gears or electrons. The team calls its biological transistor the “transcriptor.”

“Transcriptors are the key component behind amplifying genetic logic — akin to the transistor and electronics,” said Jerome Bonnet, PhD, a postdoctoral scholar in bioengineering and the paper’s lead author.

The creation of the transcriptor allows engineers to compute inside living cells to record, for instance, when cells have been exposed to certain external stimuli or environmental factors, or even to turn on and off cell reproduction as needed.

“Biological computers can be used to study and reprogram living systems, monitor environments and improve cellular therapeutics,” said Drew Endy, PhD, assistant professor of bioengineering and the paper’s senior author.

The biological computer

In electronics, a transistor controls the flow of electrons along a circuit. Similarly, in biologics, a transcriptor controls the flow of a specific protein, RNA polymerase, as it travels along a strand of DNA.

“We have repurposed a group of natural proteins, called integrases, to realize digital control over the flow of RNA polymerase along DNA, which in turn allowed us to engineer amplifying genetic logic,” said Endy.

Using transcriptors, the team has created what are known in electrical engineering as logic gates that can derive true-false answers to virtually any biochemical question that might be posed within a cell.

They refer to their transcriptor-based logic gates as “Boolean Integrase Logic,” or “BIL gates” for short.

Transcriptor-based gates alone do not constitute a computer, but they are the third and final component of a biological computer that could operate within individual living cells.

Despite their outward differences, all modern computers, from ENIAC to Apple, share three basic functions: storing, transmitting and performing logical operations on information.

Last year, Endy and his team made news in delivering the other two core components of a fully functional genetic computer. The first was a type of rewritable digital data storage within DNA. They also developed a mechanism for transmitting genetic information from cell to cell, a sort of biological Internet.

It all adds up to creating a computer inside a living cell.

Boole’s gold

Digital logic is often referred to as “Boolean logic,” after George Boole, the mathematician who proposed the system in 1854. Today, Boolean logic typically takes the form of 1s and 0s within a computer. Answer true, gate open; answer false, gate closed. Open. Closed. On. Off. 1. 0. It’s that basic. But it turns out that with just these simple tools and ways of thinking you can accomplish quite a lot.

“AND” and “OR” are just two of the most basic Boolean logic gates. An “AND” gate, for instance, is “true” when both of its inputs are true — when “a” and “b” are true. An “OR” gate, on the other hand, is true when either or both of its inputs are true.

In a biological setting, the possibilities for logic are as limitless as in electronics, Bonnet explained. “You could test whether a given cell had been exposed to any number of external stimuli — the presence of glucose and caffeine, for instance. BIL gates would allow you to make that determination and to store that information so you could easily identify those which had been exposed and which had not,” he said.

By the same token, you could tell the cell to start or stop reproducing if certain factors were present. And, by coupling BIL gates with the team’s biological Internet, it is possible to communicate genetic information from cell to cell to orchestrate the behavior of a group of cells.

“The potential applications are limited only by the imagination of the researcher,” said co-author Monica Ortiz, a PhD candidate in bioengineering who demonstrated autonomous cell-to-cell communication of DNA encoding various BIL gates.

Building a transcriptor

To create transcriptors and logic gates, the team used carefully calibrated combinations of enzymes — the integrases mentioned earlier — that control the flow of RNA polymerase along strands of DNA. If this were electronics, DNA is the wire and RNA polymerase is the electron.

“The choice of enzymes is important,” Bonnet said. “We have been careful to select enzymes that function in bacteria, fungi, plants and animals, so that bio-computers can be engineered within a variety of organisms.”

On the technical side, the transcriptor achieves a key similarity between the biological transistor and its semiconducting cousin: signal amplification.

With transcriptors, a very small change in the expression of an integrase can create a very large change in the expression of any two other genes.

To understand the importance of amplification, consider that the transistor was first conceived as a way to replace expensive, inefficient and unreliable vacuum tubes in the amplification of telephone signals for transcontinental phone calls. Electrical signals traveling along wires get weaker the farther they travel, but if you put an amplifier every so often along the way, you can relay the signal across a great distance. The same would hold in biological systems as signals get transmitted among a group of cells.

“It is a concept similar to transistor radios,” said Pakpoom Subsoontorn, a PhD candidate in bioengineering and co-author of the study who developed theoretical models to predict the behavior of BIL gates. “Relatively weak radio waves traveling through the air can get amplified into sound.”

Public-domain biotechnology

To bring the age of the biological computer to a much speedier reality, Endy and his team have contributed all of BIL gates to the public domain so that others can immediately harness and improve upon the tools.

“Most of biotechnology has not yet been imagined, let alone made true. By freely sharing important basic tools everyone can work better together,” Bonnet said.

The research was funded by the National Science Foundation and the Townshend Lamarre Foundation.

Publication: Jerome Bonnet, et al., “Amplifying Genetic Logic Gates,” Science, 28 March 2013: DOI: 10.1126/science.1232758

Source: Andrew Myers, Stanford University

Nanoparticle Immunogen Design Targets HIV and Other Fast-Mutating Viruses

The researchers demonstrated their new technique by engineering a compound that has promise to initiate an otherwise rare immune response against many types of HIV. Here, the germline-targeting immunogen eOD-GT6 (red) is shown bound to its target, the germline VRC01 antibody (magenta and yellow).

In a newly published study, researchers detail a new technique for vaccine design that offers insight on how to stimulate the immune system to produce the right kind of antibody response to protect against fast-changing viruses.

La Jolla, California – A team led by scientists from The Scripps Research Institute (TSRI) and the International AIDS Vaccine Initiative (IAVI) has unveiled a new technique for vaccine design that could be particularly useful against HIV and other fast-changing viruses.

The report, which appears March 28, 2013, in Science Express, the early online edition of the journal Science, offers a step toward solving what has been one of the central problems of modern vaccine design: how to stimulate the immune system to produce the right kind of antibody response to protect against a wide range of viral strains. The researchers demonstrated their new technique by engineering an immunogen (substance that induces immunity) that has promise to reliably initiate an otherwise rare response effective against many types of HIV.

“We’re hoping to test this immunogen soon in mice engineered to produce human antibodies, and eventually in humans,” said team leader William R. Schief, who is an associate professor of immunology and member of the IAVI Neutralizing Antibody Center at TSRI.

Seeking a Better Way

For highly variable viruses such as HIV and influenza, vaccine researchers want to elicit antibodies that protect against most or all viral strains—not just a few strains, as seasonal flu vaccines currently on the market. Vaccine researchers have identified several of these broadly neutralizing antibodies from long-term HIV-positive survivors, harvesting antibody-producing B cells from blood samples and then sifting through them to identify those that produce antibodies capable of neutralizing multiple strains of HIV. Such broadly neutralizing antibodies typically work by blocking crucial functional sites on a virus that are conserved among different strains despite high mutation elsewhere.

However, even with these powerful broadly neutralizing antibodies in hand, scientists need to find a way to elicit their production in the body through a vaccine. “For example, to elicit broadly neutralizing antibodies called VRC01-class antibodies that neutralize 90 percent of known HIV strains, you could try using the HIV envelope protein as your immunogen,” said Schief, “but you run into the problem that the envelope protein doesn’t bind with any detectable affinity to the B cells needed to launch a broadly neutralizing antibody response.”

To reliably initiate that VRC01-class antibody response, Schief and his colleagues therefore sought to develop a new method for designing vaccine immunogens.

From Weak to Strong

Joseph Jardine, a TSRI graduate student in the Schief laboratory, evaluated the genes of VRC01-producing B cells in order to deduce the identities of the less mature B cells—known as germline B cells—from which they originate. Germline B cells are major targets of modern viral vaccines, because it is the initial stimulation of these B cells and their antibodies that leads to a long-term antibody response.

In response to vaccination, germline B cells could, in principle, mature into the desired VRC01-producing B cells—but natural HIV proteins fail to bind or stimulate these germline B cells so they cannot get the process started. The team thus set out to design an artificial immunogen that would be successful at achieving this.

Jardine used a protein modeling software suite called Rosetta to improve the binding of VRC01 germline B cell antibodies to HIV’s envelope protein. “We asked Rosetta to look for mutations on the side of the HIV envelope protein that would help it bind tightly to our germline antibodies,” he said.

Rosetta identified dozens of mutations that could help improve binding to germline antibodies. Jardine then generated libraries that contained all possible combinations of beneficial mutations, resulting in millions of mutants, and screened them using techniques called yeast surface display and FACS. This combination of computational prediction and directed evolution successfully produced a few mutant envelope proteins with high affinity for germline VRC01-class antibodies.

Jardine then focused on making a minimal immunogen—much smaller than HIV envelope—and so continued development using the “engineered outer domain (eOD)” previously developed by Po-Ssu Huang in the Schief lab while Schief was at the University of Washington. Several iterative rounds of design and selection using a panel of germline antibodies produced a final, optimized immunogen—a construct they called eOD-GT6.

A Closer Look

To get a better look at eOD-GT6 and its interaction with germline antibodies, the team turned to the laboratory of Ian A. Wilson, chair of the Department of Integrative Structural and Computational Biology and a member of the IAVI Neutralizing Antibody Center at TSRI.

Jean-Philippe Julien, a senior research associate in the Wilson laboratory, determined the 3D atomic structure of the designed immunogen using X-ray crystallography—and, in an unusual feat, also determined the crystal structure of a germline VRC01 antibody, plus the structure of the immunogen and antibody bound together.

“We wanted to know whether eOD-GT6 looked the way we anticipated and whether it bound to the antibody in the way that we predicted—and in both cases the answer was ‘yes’,” said Julien. “We also were able to identify the key mutations that conferred its reactivity with germline VRC01 antibodies.”

Mimicking a Virus

Vaccine researchers know that such an immunogen typically does better at stimulating an antibody response when it is presented not as a single copy but in a closely spaced cluster of multiple copies, and with only its antibody-binding end exposed. “We wanted it to look like a virus,” said Sergey Menis, a visiting graduate student in the Schief laboratory.

Menis therefore devised a tiny virus-mimicking particle made from 60 copies of an obscure bacterial enzyme and coated it with 60 copies of eOD-GT6. The particle worked well at activating VRC01 germline B cells and even mature B cells in the lab dish, whereas single-copy eOD-GT6 did not.

“Essentially it’s a self-assembling nanoparticle that presents the immunogen in a properly oriented way,” Menis said. “We’re hoping that this approach can be used not just for an HIV vaccine but for many other vaccines, too.”

The next step for the eOD-GT6 immunogen project, said Schief, is to test its ability to stimulate an antibody response in lab animals that are themselves engineered to produce human germline antibodies. The difficulty with testing immunogens that target human germline antibodies is that animals typically used for vaccine testing cannot make those same antibodies. So the team is collaborating with other researchers who are engineering mice to produce human germline antibodies. After that, he hopes to learn how to drive the response, from the activation of the germline B cells all the way to the production of mature, broadly neutralizing VRC01-class antibodies, using a series of designed immunogens.

Schief also hopes they will be able to test their germline-targeting approach in humans sooner rather than later, noting “it will be really important to find out if this works in a human being.”

The first authors of the paper, “Rational HIV immunogen design to target specific germline B cell receptors,” were Jardine, Julien and Menis. Co-authors were Takayuki Ota and Devin Sok of the Nemazee and Burton laboratories at TSRI, respectively; Travis Nieusma of the Ward laboratory at TSRI; John Mathison of the Ulevitch laboratory at TSRI; Oleksandr Kalyuzhniy and Skye MacPherson, researchers in the Schief laboratory from IAVI and TSRI, respectively; Po-Ssu Huang and David Baker of the University of Washington, Seattle; Andrew McGuire and Leonidas Stamatatos of the Seattle Biomedical Research Institute; and TSRI principal investigators Andrew B. Ward, David Nemazee, Ian A. Wilson, and Dennis R. Burton, who is also head of the IAVI Neutralizing Center at TSRI.

The project was funded in part by IAVI; the National Institutes of Health (AI84817, AI081625 and AI33292); and the Ragon Institute.

Publication: Joseph Jardine, et al., “Rational HIV Immunogen Design to Target Specific Germline B Cell Receptors,” 2013, Science DOI: 10.1126/science.1234150

Source: The Scripps Research Institute

Image: The Scripps Research Institute

Single Antibody is Broadly Effective Against a Variety of Human Cancer Tumors

Single Antibody is Broadly Effective Against a Variety of Human Cancer Tumors

In a new study, a team of scientists have shown that a single antibody shrinks variety of human tumors transplanted into mice, finding that anti-CD47 antibodies can dramatically inhibit the growth of human solid tumors by blocking the ability of CD47 to transmit the ‘don’t-eat-me’ signal to macrophages.

Human tumors transplanted into laboratory mice disappeared or shrank when scientists treated the animals with a single antibody, according to a new study from the Stanford University School of Medicine. The antibody works by masking a protein flag on cancer cells that protects them from macrophages and other cells in the immune system. The scientists achieved the findings with human breast, ovarian, colon, bladder, brain, liver and prostate cancer samples.

It is the first antibody treatment shown to be broadly effective against a variety of human solid tumors, and the dramatic response — including some overt cures in the laboratory animals — has the investigators eager to begin phase-1 and –2 human clinical trials within the next two years.

“Blocking this ‘don’t-eat-me’ signal inhibits the growth in mice of nearly every human cancer we tested, with minimal toxicity,” said professor of pathology Irving Weissman, MD, who directs Stanford’s Institute of Stem Cell Biology and Regenerative Medicine and the Ludwig Center for Cancer Stem Cell Research and Medicine at Stanford. “This shows conclusively that this protein, CD47, is a legitimate and promising target for human cancer therapy.”

The antibody treatment also significantly inhibited the ability of the tumors to metastasize throughout the animals’ bodies.

“This is exciting work and will surely trigger a worldwide wave of research designed to convert this strategy into useful therapies,” said Robert Weinberg, PhD, a professor of biology at the Whitehead Institute for Biomedical Research in Massachusetts who was not involved in the research. “Mobilizing the immune system to attack solid tumors has been a longstanding goal of many cancer researchers for decades.”

The research was published online March 26 in the Proceedings of the National Academy of Sciences. Weissman, who is the Virginia & D.K. Ludwig Professor for Clinical Investigation in Cancer Research at Stanford and a member of the Stanford Cancer Institute, is the senior author of the research. Postdoctoral scholars Stephen Willingham, PhD, and Jens-Peter Volkmer, MD, are the co-first authors of the study.

Previous work in Weissman’s lab has shown that CD47 is normally expressed on the surfaces of circulating blood stem cells to protect them from immune cells called macrophages. Macrophages patrol the body looking for signs of trouble in the form of invaders or rogue cells, but they sometimes latch onto the wrong targets. CD47 prompts them to release cells they’ve grabbed by mistake.

Weissman and his colleagues also showed previously that some types of cancer cells — particularly those of blood cancers such as leukemia and lymphoma — have figured out a way to game the system and use this “don’t-eat-me signal” to their advantage by expressing CD47 on their own surfaces. In 2010, they found that blocking CD47 with a specific antibody (plus adding another to further stimulate the macrophages’ killing instinct) can cure some cases of human non-Hodgkin’s lymphoma in mice. But it wasn’t known until now how widespread or clinically important the phenomenon would be in human solid tumors.

In the current study, Willingham and Volkmer collected surgical samples of a variety of human tumors, including ovarian, breast, colon, bladder, brain, liver and prostate. To do so, they enlisted the help of clinical experts from across the School of Medicine, including those specializing in oncology, urology, obstetrics and gynecology, radiation oncology, neurosurgery, hematology, pathology, otolaryngology and hepatology.

They showed that nearly every human cancer cell they examined expressed CD47 — usually at higher levels (on average, about three times more) than did non-cancerous cells. Furthermore, people whose cancer cells express a lot of CD47 tend to have shorter life spans than people with similar cancers that express less CD47. This suggests that an analysis of the levels of CD47 expression in some types of tumors could be a valuable prognostic tool for patients and their doctors.

Willingham and Volkmer then implanted the different human tumor cells into matching locations in the bodies of mice — breast cancer tumors into the mammary fat pads, and ovarian cancer tumors into the abdomen, for example. Once the tumors were well-established (after two weeks or more), they treated the animals with the anti-CD47 antibody.

The researchers saw that most of the established tumors begin to shrink and even, in some cases, disappear within weeks of treatment with the antibody. In one case, antibody treatment cured five mice injected with the same human breast cancer cells. When the tumor was gone, the treatment was discontinued; the mice were monitored for four months with no signs of recurrence.

“These results indicate that anti-CD47 antibodies can dramatically inhibit the growth of human solid tumors by blocking the ability of CD47 to transmit the ‘don’t-eat-me’ signal to macrophages,” concluded the authors.

“If the tumor was highly aggressive,” said Weissman, “the antibody also blocked metastasis. It’s becoming very clear that, in order for a cancer to survive in the body, it has to find some way to evade the cells of the innate immune system.” The innate immune system is the body’s first line of defense against pathogens like bacteria and viruses. Unlike the adaptive immunity conferred by antibodies and T cells that recognize and battle specific molecules, cells of the innate immune system, like macrophages, respond non-specifically to a variety of threats.

The researchers’ approach didn’t work in every animal, though. A set of mice with breast cancer cells from one human patient experienced no benefit from antibody treatment. “There’s certainly more to learn,” said Weissman. “We need to learn more about the relationship between macrophages and tumor cells, and how to draw more macrophages to the tumors.” He suggested that reducing the size of a tumor with surgery or radiotherapy before antibody treatment could make the treatment more effective. Another option, he added, would be to use a second antibody in addition to CD47 that would further stimulate the ability of the macrophages or other immune cells to kill the cancer cells.

While treatment modifications may be beneficial, the findings about the effect of the single antibody are promising in their own right and set the stage for advancing the research. “We believe these results show that we should move forward quickly but cautiously into human clinical trials for many types of solid tumors,” Weissman said.

In addition to Willingham and Volkmer, other Stanford scientists involved in the research include postdoctoral scholars Andrew Gentles, PhD, Ferenc Scheeren, PhD, Anne Volkmer, MD, Chris Sun, PhD, Oihana Murillo, PhD, Maddalena Adorno, PhD, and Tal Raveh, PhD; graduate students Badreddin Edris, Pradeep Rajendran and Adriel Cha; medical or MSTP students Kipp Weiskopf, Chad Tang and Diane Tseng; life science research assistants Tejaswitha Naik, Theresa Storm, Humberto Contreras-Trujillo, Justin Cohen and Adriane Mosley; instructors Debashis Sahoo, PhD, and Piero Dalerba, MD; senior research scientists Siddhartha Mitra, PhD, and Mei-Sze Chua, PhD; clinical fellow Jian Wang, MD, PhD; CIRM research scholar Robin Martin; research and development engineer Patricia Lovelace; research associate Dongkyoon Kim, PhD; intern Mark Chao, MD, PhD; resident Siddhartha Jaiswal, MD, PhD; assistant professors Robert Chin, MD, PhD, Gordon Li, MD, Ravindra Majeti, MD, PhD, John Sunwoo, MD, and Ash Alizadeh, MD, PhD; associate professor Nelson Teng, MD, PhD; professors Samuel So, MD, Matt van de Rijn, MD, PhD, and Griffith Harsh, MD; associate director of the Stanford stem cell institute Michael Clarke, MD; and neurosurgery chair Gary Steinberg, MD, PhD.

This work was supported by the Joseph & Laurie Lacob Gynecologic/Ovarian Cancer Fund, the Jim & Carolyn Pride Fund, the Virginia & D.K. Ludwig Fund for Cancer Research, the Weston Havens Foundation, the National Cancer Institute, the Department of Defense, the California Institute for Regenerative Medicine and anonymous donors.

Publication: Stephen B. Willingham, et al., “The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors,” PNAS April 24, 2012 vol. 109 no. 17 6662-6667; doi: 10.1073/pnas.1121623109

Source: Krista Conger, Stanford University

Self-help programs may help combat childhood obesity

Self-help programs may help combat childhood obesity

Washington: Overweight children and their parents can shed extra pounds on their own, if they follow a self-help treatment program under the guidance of medical experts, according to a new study.

Kerri Boutelle, PhD, associate professor of pediatrics and psychiatry at UC San Diego School of Medicine and colleagues enrolled 50 overweight or obese children between the ages of 8 and 12 and their family members in a low-intensity, 5-month long treatment for childhood obesity, measuring the effects on a child’s weight (measured as body mass index or BMI) immediately post-treatment and six months later.

Self-help programs may help combat childhood obesity

The researchers also evaluated whether the intervention promoted improvements in eating behavior and physical activity among children and parents. The results of the guided, self-help intervention program showed a significant decrease in BMI immediately after completing the 5-month treatment, losses that were maintained six months later.

According to the UCSD researchers, such a program may be an improvement over current methods, especially because the program is designed to fit a busy family’s schedule.

“The guided self-help treatment includes offering structure along with a self-help program to help families stick to the program,” said Boutelle. “Parents and their children are given a manual, and each week they read a chapter and try to apply the skills at home. Every other week they come in to our clinic at UC San Diego School of Medicine for 20 minutes and discuss how things were going with an interventionist. This is very different than traditional weight control programs where parents and kids come in every week for an hour-and-a-half-long group-based program.”

“Importantly, the initial results of this study showed that that a self-help program, guided by professionals, may be as effective in helping kids to lose weight as a traditional, clinic-based weight loss program,” Boutelle concluded.

The study was recently published in the journal Pediatrics.