Caltech Bioethics Forum: HeLa Cells in the Lab

Henrietta Lacks died of cervical cancer in 1951 and, ever since, samples of her uniquely immortal cancerous cells have been used in scientific research, sparking great leaps in medical knowledge.

But the cells—taken without her knowledge or consent—have also fueled controversy and called into question the ethical underpinnings of the research they made possible. Her story, made famous in the book, The Immortal Life of Henrietta Lacks, by science writer Rebecca Skloot, underscores the continuing need—65 years after her death—for society to find a way to balance the advancement of medical knowledge with the protection of individual rights.

At a February 22 bioethics forum at Baxter Lecture Hall, Caltech president Thomas F. Rosenbaum introduced a panel of Caltech faculty that examined the ethics of using Lacks's cells—known as HeLa cells—along with issues of privacy, informed consent, and who profits from the technologies her cells engendered. Caltech trustee Ronald L. Olson moderated the panel of Caltech faculty, which featured David Baltimore, President Emeritus and the Robert Andrews Millikan Professor of Biology; Ellen Rothenberg, the Albert Billings Ruddock Professor of Biology; Barbara J. Wold, the Bren Professor of Molecular Biology; and Changhuei Yang, professor of electrical engineering, bioengineering, and medical engineering.

The evening event revisited a topic that incoming freshmen had tackled earlier in the academic year in roundtable discussions of the book, which the students had been asked to read prior to their arrival at Caltech.

"HeLa cells were a miracle," said Baltimore, who has used them in his research since 1962. After noting their incalculable value—and how rare it was to have found cells that underwent the specific mutations that conferred their ability to divide indefinitely in culture—he brought the discussion back to the question of who owns the cells.

"So it seems to me the question is, what rights does she have as a consequence of this rare, basically random event, that made her cells different than anybody else's cells? . . . We are the product of a genetic lottery. What rights do we get as a consequence of our particular genes? Everyone else around us has a representation of those same genes, but not identical. What is ownership in this case?"

Rothenberg discussed how the cells have enabled key advances in molecular biology, stem cell research, and immunology—advances that would have been considered "complete science fiction fantasy" when Lacks was alive. Because the innovations her cells made possible would have been impossible to foresee, questions naturally arise as to whether Lacks could have understood the ramifications of her consent, had it been sought.

The structure of DNA was only discovered two years after Lacks' death, and the revolution in molecular biology that followed completely transformed the possibilities for use of any human cells that were able to grow in culture. "How could she possibly have given informed consent? In the case of a rapidly advancing field like molecular biology, there's no way she could have been asked at that time what she was really consenting to," Rothenberg said.

When the conversation returned to ownership of a patient's cells, and who should profit from their use, Rothenberg pointed out that simply saying the patient owns them, period, could generate a raft of unintended consequences. For example, medical institutions might, for legal liability reasons, refuse to accept certain tissue samples, hampering the delivery of personalized medicine to patients. Equally disturbing, she says, would be the possibility that the commercially valuable products of cells might incentivize individuals to view a cancer-patient relative "as a possible cash cow, and sell their tissues in the hopes of winning the lottery. . . . You definitely don't want people to be in a position where they or their family members want to sell parts of their body because they're starving."

Wold said her research seldom involves HeLa cells but, she added, "that doesn't mean I'm not an avid consumer of what's been learned over 60 years of studying them." She hailed the "beautiful science" the cells have engendered, but lamented the scientific community's repeated failings in communicating with and involving the Lacks family over the years as to how the cells were being used and what was being learned from them. For example, she said, teams of researchers in Germany and at the University of Washington sequenced and published the HeLa genome in 2013 and made the information freely available worldwide. In doing so, however, they made portions of the family's genome public—without thinking to seek the family's approval or tell the family what was happening. The research community "quickly recognized this as a catastrophe," said Wold, prompting the creation of a board that includes Lacks family members and now regulates access to the data.

Such ethical considerations continued during the event's Q&A, which stirred discussion about such critical questions as how to address medical privacy when one family member's consent might make public another family member's information, and whether proposed consent rules might jeopardize access to older cell lines that were obtained prior to a stricter consent regime.

Yang, whose lab has used HeLa cells since 2008, said he only recently learned about the ethical concerns around their provenance, adding, "Honestly I was quite surprised to find there were all these [controversies]. . . . As an outsider to American culture—I actually grew up in Singapore—my instinct would be that the DNA is a common good, not personal property. If my cells would be useful for research, I would gladly give them up without any expectations."

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Caltech Bioethics Forum: HeLa Cells in the Lab
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Caltech faculty examine the ethics of using Henrietta Lacks’s cells—covering issues of privacy, informed consent, and who profits from the technologies her cells engendered.
Friday, April 15, 2016
Beckman Institute Auditorium – Beckman Institute

A Celebration of Eric Davidson: Visionary Insights into the Genomic Control of Development and Evolution

Gradinaru and Benardini Receive Presidential Early Career Awards

Viviana Gradinaru (BS '05), an assistant professor of biology and biological engineering, and James Benardini, a planetary protection engineer at JPL, have been named as recipients of the 2016 Presidential Early Career Award for Scientists and Engineers (PECASE).

The PECASE awards were created to foster innovative developments in science and technology, increase awareness of careers in science and engineering, give recognition to the scientific missions of participating agencies, enhance connections between fundamental research and many of the grand challenges facing the nation, and highlight the importance of science and technology for America's future. The award is the highest honor bestowed by the United States government on science and engineering professionals in the early stages of their independent research careers.

Viviana Gradinaru is an assistant professor of biology and biological engineering as well as the faculty director of the Beckman Institute Center for CLARITY, Optogenetics, and Vector Engineering Research (CLOVER). Her work focuses on developing technologies such as optogenetics (using light to control genetically modified cells) and tissue clearing (that can render rodent tissues and bodies transparent via PARS CLARITY). Most recently, she and her team have discovered how to modify the protein shell of a harmless virus to successfully enter the adult mouse brain from the bloodstream—crossing the so-called blood-brain barrier—and deliver genes to cells of the nervous system. Gradinaru employs these technologies for mapping and modulating brain networks to understand and develop therapies for neurological diseases.

James (Nick) Benardini is the planetary protection lead on the InSight and Mars 2020 missions. He and his colleagues study how to minimize microbial and other biological contamination on outgoing space missions. This involves the use of clean rooms and microbial reduction modalities in addition to looking for genetic traces on samples collected from spacecraft and spacecraft-associated surfaces.

In addition to Gradinaru, three other Caltech alumni were named as 2016 PECASE recipients: Alon Gorodetsky (PhD '09), Jon Simon (BS '04), and Tammy Ma (BS '05).

The winners will receive their awards at a ceremony in Washington, D.C., this spring.

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Gradinaru and Benardini Receive Presidential Early Career Awards
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Viviana Gradinaru and James Benardini have been named as recipients of the 2016 Presidential Early Career Award for Scientists and Engineers.
Thursday, March 17, 2016
Beckman Institute Auditorium – Beckman Institute

New Horizons in Biology 2016 Faculty Hiring Symposium

Studying Memory's 'Ripples'

Caltech neuroscientists have looked inside brain cells as they undergo the intense bursts of neural activity known as "ripples" that are thought to underlie memory formation. 

During ripples, a small fraction of brain cells, or neurons, fire synchronously in area CA1, a part of the hippocampus that is thought to be an important relay station for memories. "During a ripple, about 10 percent of the neurons in CA1 are activated, and these active neurons all fire within a tenth of a second," says Caltech graduate student Brad Hulse. "Two big questions have been: How do the remaining 90 percent of CA1 neurons stay quiet? And what is synchronizing the firing of the active neurons?"

In a new study, published online on February 17 in the journal Neuron, Hulse and his colleagues used a novel approach to show how the combination of excitatory and inhibitory inputs to CA1 work together to synchronize the firing of active neurons while keeping most neurons silent during ripples.

"For a long time, people studied these events by placing an electrode outside of a cluster of neurons. These extracellular recordings can tell you about the output of a group of brain cells, but they tell you very little about the inputs they're receiving," says study coauthor and Caltech research scientist Evgueniy Lubenov.

The Caltech scientists combined extracellular recording with a technique to look inside a neuron during ripples. They used fine glass pipettes with tips thinner than a tenth of the width of a human hair to measure directly the voltage difference, or "electrical potential," across the cellular membrane of individual neurons in awake mice.

Employing these two techniques in tandem allowed the scientists to monitor the activity inside a single neuron while still observing how the larger network was behaving. This in turn enabled them to piece together how excitatory inputs from CA3, a hippocampal region where memories are formed, affect the output of brain cells called pyramidal neurons in CA1. These neurons are important for transferring newly coded memories to other brain areas such as the neocortex for safekeeping and long-term storage. Ripples are thought to be the mechanism by which this transfer occurs.

The team discovered that the membrane potential of CA1 pyramidal cells increases during ripples. Surprisingly, this increase is relatively constant and independent of the strength of the input from CA3. For this to be the case, the direct excitation from CA3 must be balanced by proportional inhibition. The source of this inhibition is presumed to be a class of brain cells called feedforward interneurons, which receive direct inputs from CA3 and inhibit CA1 pyramidal cells.

"There seems to be a circuit mechanism that balances excitation and inhibition, so that for most neurons, these two forces cancel out," says study leader Thanos Siapas, professor of computation and neural systems at Caltech.

Without a balanced inhibition, all of the neurons in CA1 could fire when the excitatory input is large enough. "This could cause runaway excitation and possibly trigger a seizure," says Hulse, who is the first author of the new study.

The team's finding explains why most CA1 pyramidal neurons remain silent during ripples, but it raises two important questions: Why do any neurons fire at all? And what controls the precise timing of those that do fire?

The Caltech researchers found that active neurons receive a much stronger excitatory input from CA3 than silent neurons do—one that is large enough to overcome the balancing inhibition. This large excitation originates from CA3 neurons with particularly strong connections to the active CA1 neurons. These connections are believed to be modified during behavior to encode memories.

Hence, it is the specific identity of CA3 neurons, rather than their sheer number, that is responsible for making CA1 neurons fire, the researchers say. This system might seem overly complex and redundant, but the end result is a flexible circuit—an ever-changing mosaic of active and inactive pyramidal neurons. "It's a shifting mosaic, but it's one that is dependent on the organism's memories and experience," Siapas says.

How do ripples exert their influence on the rest of the brain? The membrane potential of each neuron oscillates very rapidly during ripples to synchronize the firing of cells to within a few thousandths of a second. "By coordinating their activities, the CA1 neurons are maximizing the impact of their output on downstream areas of the brain. The overall effect is more powerful than if each neuron fired independently," Lubenov says. "It is the difference between clapping independently or in unison with others at a concert. The effect in the latter case is stronger, even with the same number of people applauding."

Neuroscientists previously thought that these fast oscillations were generated by rhythmic firing of inhibitory neurons, but the Caltech team showed that this cannot be the whole story. "Our experiments suggest that it is the interplay between rhythmic excitation and inhibition that drives these fast oscillations," Hulse says.

The paper, "Membrane Potential Dynamics of CA1 Pyramidal Neurons during Hippocampal Ripples in Awake Mice," is also coauthored by Laurent C. Moreaux, a research scientist at Caltech. Funding for the work was provided by the Mathers Foundation, the Gordon and Betty Moore Foundation, the National Institutes of Health, and the National Science Foundation. 

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Studying Memory's 'Ripples'
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Neuroscientists look inside brain cells undergoing the bursts of activity, or "ripples," that underlie memory formation

Caltech Biologists Identify Gene That Helps Regulate Sleep

Caltech biologists have performed the first large-scale screening in a vertebrate animal for genes that regulate sleep, and have identified a gene that when overactivated causes severe insomnia. Expression of the gene, neuromedin U (Nmu), also seems to serve as nature's stimulant—fish lacking the gene take longer to wake up in the morning and are less active during the day.

The findings improve our understanding of how sleep is regulated—a process that we know surprisingly little about despite its clear importance. In the long term, the results suggest Nmu as a potential candidate for new therapies to address sleep disorders.

A paper describing the new screening process and its results appears in the February 17, 2016, issue of the journal Neuron. David Prober, assistant professor of biology at Caltech, began the work as a postdoctoral fellow at Harvard University, and has continued the work at Caltech since 2009. The lead authors on the paper are Cindy Chiu (PhD '14), a former graduate student in Prober's lab, and Jason Rihel, who collaborated with Prober at Harvard and now has his own lab at University College London.

"Sleep is a mysterious process," says Prober. "We spend a third of our lives doing it, and every animal with a complex nervous system seems to do it, so it must be important. But we still don't understand why we do it or how it's regulated."

Genetic screens are a powerful method that can help identify the genetic basis of such behaviors. They typically involve mutating the DNA of thousands of animals, raising them, identifying any resulting physical or behavioral differences, and determining which altered gene produced each mutation. This approach works well for simple model organisms, such as fruit flies and worms, because their anatomy is relatively simple and it is easy to raise large numbers of them, but is far more difficult in vertebrates, such as rodents.

Recently, zebrafish have emerged as a valuable vertebrate model system for studying sleep. Compared to a mouse, the small, striped fish are much easier to work with. Many can be raised in a small space (a larva is about 4 millimeters long, about the same size as a fruit fly); they develop quickly, exhibiting complex behaviors, such as hunting, by the time they are five days old; and they are transparent during their embryonic and larval stages, making it simpler for researchers to track what is happening inside their brains. Like humans, zebrafish sleep for consolidated periods of time at night. Furthermore, Prober says, "anatomical and molecular similarities between zebrafish and mammalian brains suggest that the basic neural circuits regulating sleep in zebrafish are likely conserved in mammals."

Rather than mutating the DNA and testing which functions were lost, the researchers used a gain-of-function approach in the new study. Just after fertilization, when the zebrafish embryos were still single cells, the researchers injected them with a DNA molecule, called a plasmid, carrying a gene that was inserted into the genome of some of the cells in each fish. In particular, they wanted to test genes that are predicted to encode for secreted proteins—those, like neuropeptides, that cells make and then release. Many of the genes that have been identified as being involved in sleep encode neuropeptides.

Using a genetic switch called a heat-shock promoter, which turns on only when the fish are heated to about 37 degrees Celsius, the biologists were able to control when the fish expressed each inserted gene. They kept the switch on long enough for the fish to overexpress each gene, making many copies of the products. Then they used computerized video trackers to monitor the fish for several days to see which genes affected sleep.

Next, the researchers made transgenic zebrafish for each of the genes that had demonstrated strong effects on sleep in the genetic screen. That labor-intensive approach gave them zebrafish in which all cells overexpressed a particular gene in response to heat shock, providing more robust results.

In the end, the most significant change resulted from overexpression of Nmu, a gene that is also found in mammals and is expressed in a part of the brain called the hypothalamus.

"After heat shock, the fish that overexpress Nmu are much more active both during the day and at night," says Prober. "The fish almost don't sleep at all the night following the heat shock—so they display a very profound form of insomnia."

When the researchers mutated the zebrafish so that they did not have Nmu, the larvae were less active during the day. Adult fish without the gene were particularly sluggish first thing in the morning.

Like humans, zebrafish normally start to wake up at the end of the night and then become much more active when the lights turn on. "The fish without Nmu are defective in this anticipation of dawn," says Prober. "So it seems that this gene is particularly important for the transition from nighttime sleep to daytime wakefulness."

To explore how Nmu promotes wakefulness, the researchers first investigated the gene's role in a stress response pathway known as the hypothalamic-pituitary-adrenal (HPA) axis. Researchers had previously shown Nmu to be involved in arousal caused by stressful situations and hypothesized that it was involved in activating the HPA axis. However, Prober and his colleagues found that Nmu suppressed sleep to the same extent in zebrafish mutants lacking a protein called the glucocorticoid receptor, which is necessary for HPA axis signaling, as it did in fish with a functional glucocorticoid receptor, suggesting that the gene does not act through the HPA axis.

The researchers then went back to the drawing board and asked which neurons in the brain became activated as a result of Nmu overexpression. Using a technique that labels activated neurons, they saw strong activation of a handful of cells that express a gene called corticotrophin-releasing hormone (CRH) in the brainstem.

"That was surprising because CRH is the gene that initiates the HPA axis response, but the cells that do that are in the hypothalamus, a different part of the brain, and they aren't activated when we overexpress Nmu," says Prober. "It's another population of CRH cells in the brainstem that are activated by Nmu overexpression."

A low dose of a drug that blocks CRH signaling completely blocked the wake-promoting effect of Nmu overexpression in zebrafish, the researchers found, whereas a higher dose also reduced wakefulness in normal fish.

"So not only is CRH signaling required for the effects of Nmu on behavior, it's also required for normal levels of activity," explains Prober.

Several wake-promoting or sleep-promoting genes and neurons have been identified, he notes. However, scientists still do not know which are the relevant ones for causing sleep disorders in humans. "Our study suggests that Nmu could be a good gene to look into."

Additional Caltech authors on the paper, "A Zebrafish Genetic Screen Identifies Neuromedin U as a Regulator of Sleep/Wake States," are Daniel A. Lee, Chanpreet Singh, Eric A. Mosser, Shijia Chen, Viveca Sapin, Uyen Pham, Jae Engle, Brett J. Niles, Christin J. Montz, and Sridhara Chakravarthy. Steven Zimmerman and Alexander F. Schier are additional authors from Harvard University. Kourosh Salehi-Ashtiani and Marc Vidal are authors from Harvard Medical School. The work was supported by grants from the National Institutes of Health, the European Research Council, University College London, the High-Tech Fund of the Dana Farber Cancer Institute, the Ellison Foundation, the Edward Mallinckrodt, Jr. Foundation, the Rita Allen Foundation, and the Brain and Behavior Research Foundation.

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Kimm Fesenmaier
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A Gene That Helps Regulate Sleep
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By conducting a genetic screen in zebrafish, biologists have identified a gene that seems to serve as nature's stimulant.
Monday, February 29, 2016

Modeling molecules at the microscale

Geobiologist Honored by National Academy of Sciences

Dianne Newman, professor of biology and geobiology at Caltech and an investigator with the Howard Hughes Medical Institute, has been awarded the National Academy of Sciences (NAS) Award in Molecular Biology for her "discovery of microbial mechanisms underlying geologic processes." The award citation recognizes her for "launching the field of molecular geomicrobiology" and fostering greater awareness of the important roles microorganisms have played and continue to play in how Earth evolved.

"Trust me, no one was more shocked than I was by this news," says Newman. "It really honors the many the exceptional people who have come through my lab over the years, as well as the geobiology field more broadly. Geobiology is a venerable old field, which offers many fascinating and important problems that would benefit from the attention of individuals trained in mechanistic research. Hopefully this award will encourage more young people from molecular and cellular biology to enter the field."

Newman's research focuses on the relationship between microorganisms and geologic processes. She has demonstrated that some bacteria in iron-rich environments, such soils and sediments, can utilize extracellular iron as a dump site for excess electrons by generating extracellular electron shuttles, including a class of metabolites formerly considered to be redox-active antibiotics. Newman has also made contributions to our understanding of other microbial metabolic processes of geological significance, including how microbes respire using arsenate instead of oxygen, and how they perform photosynthesis using iron rather than water. In addition, she and her coworkers have studied the mechanisms by which certain microbes make stromatolites and magnetosomes, two types of structures that leave biosignatures in ancient rocks. Perhaps most importantly, her team has demonstrated the power of applying genetic analysis to diverse organisms from iron-rich environments, paving the way for others to do the same.

Newman is now hoping to bring tools commonly used in geochemistry to facilitate environmentally-informed studies of pathogens in chronic infections. For example, in collaboration with Caltech professor of geobiology Alex Sessions and researchers at Children's Hospital Los Angeles, Newman's group has characterized the composition and growth rate of pathogens in mucus collecting in the lungs of individuals with cystic fibrosis. Using this information, her lab is designing new experiments to reveal the survival mechanisms utilized by microorganisms—such as Pseudomonas aeruginosa, an opportunistic bacterium that colonizes the lungs of these patients—in this environment.

The NAS Award in Molecular Biology was first given in 1962. It is presented with a medal and a $25,000 prize. Newman will receive the award on May 1, 2016, during the National Academy of Sciences' annual meeting in Washington, D.C.

Previous recipients of the award include David Baltimore, Caltech President Emeritus and the Robert Andrews Millikan Professor of Biology.

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Dianne Newman has been awarded the National Academy of Sciences Award in Molecular Biology.

Delivering Genes Across the Blood-Brain Barrier

Caltech biologists have modified a harmless virus in such a way that it can successfully enter the adult mouse brain through the bloodstream and deliver genes to cells of the nervous system. The virus could help researchers map the intricacies of the brain and holds promise for the delivery of novel therapeutics to address diseases such as Alzheimer's and Huntington's. In addition, the screening approach the researchers developed to identify the virus could be used to make additional vectors capable of targeting cells in other organs.

"By figuring out a way to get genes across the blood-brain barrier, we are able to deliver them throughout the adult brain with high efficiency," says Ben Deverman, a senior research scientist at Caltech and lead author of a paper describing the work in the February 1 online publication of the journal Nature Biotechnology.

The blood-brain barrier allows the body to keep pathogens and potentially harmful chemicals circulating in the blood from entering the brain and spinal cord. The semi-permeable blockade, composed of tightly packed cells, is crucial for maintaining a controlled environment to allow the central nervous system to function properly. However, the barrier also makes it nearly impossible for many drugs and other molecules to be delivered to the brain via the bloodstream.

To sneak genes past the blood-brain barrier, the Caltech researchers used a new variant of a small, harmless virus called an adeno-associated virus (AAV). Over the past two decades, researchers have used various AAVs as vehicles to transport specific genes into the nuclei of cells; once there, the genes can be expressed, or translated, from DNA into proteins. In some applications, the AAVs carry functional copies of genes to replace mutated forms present in individuals with genetic diseases. In other applications, they are used to deliver genes that provide instructions for generating molecules such as antibodies or fluorescent proteins that help researchers study, identify, and track certain cells.

Largely because of the blood-brain barrier problem, scientists have had only limited success delivering AAVs and their genetic cargo to the central nervous system. In general, they have relied on surgical injections, which deliver high concentrations of the virus at the injection site but little to the outlying areas. Such injections are also quite invasive. "One has to drill a hole through skull, then pierce tissue with a needle to the injection site," explains Viviana Gradinaru (BS '05), assistant professor of biology and biological engineering at Caltech and senior author on the paper. "The deeper the injection, the higher the risk of hemorrhage. With systemic injection, using the bloodstream, none of that damage happens, and the delivery is more uniform."

In addition, Gradinaru notes, "many disorders are not tightly localized. Neurodegenerative disorders like Huntington's disease affect very large brain areas. Also, many complex behaviors are mediated by distributed interacting networks. Our ability to target those networks is key in terms of our efforts to understand what those pathways are doing and how to improve them when they are not working well."

In 2009, a group led by Brian Kaspar of Ohio State University published a paper, also in Nature Biotechnology, showing that an AAV strain called AAV9 injected into the bloodstream could make its way into the brain—but it was only efficient when used in neonatal, or infant, mice.

"The big challenge was how do we achieve the same efficiency in an adult," says Gradinaru.

Although one might like to design an AAV that is up to the task, the number of variables that dictate the behavior of any given virus, as well as the intricacies of the brain and its barrier, make that extremely challenging. Instead, the researchers developed a high-throughput selection assay, CREATE (Cre REcombinase-based AAV Targeted Evolution), that allowed them to test millions of viruses in vivo simultaneously and to identify those that were best at entering the brain and delivering genes to a specific class of brain cells known as astrocytes.

They started with the AAV9 virus and modified a gene fragment that codes for a small loop on the surface of the capsid—the protein shell of the virus that envelops all of the virus' genetic material. Using a common amplification technique, known as polymerase chain reaction (PCR), they created millions of viral variants. Each variant carried within it the genetic instructions to produce more capsids like itself.

Then they used their novel selection process to determine which variants most effectively delivered genes to astrocytes in the brain. Importantly, the new process relies on strategically positioning the gene encoding the capsid variants on the DNA strand between two short sequences of DNA, known as lox sites. These sites are recognized by an enzyme called Cre recombinase, which binds to them and inverts the genetic sequence between them. By injecting the modified viruses into transgenic mice that only express Cre recombinase in astrocytes, the researchers knew that any sequences flagged by the lox site inversion had successfully transferred their genetic cargo to the target cell type—here, astrocytes.

After one week, the researchers isolated DNA from brain and spinal cord tissue, and amplified the flagged sequences, thereby recovering only the variants that had entered astrocytes.

Next, they took those sequences and inserted them back into the modified viral genome to create a new library that could be injected into the same type of transgenic mice. After only two such rounds of injection and amplification, a handful of variants emerged as those that were best at crossing the blood-brain barrier and entering astrocytes.

"We went from millions of viruses to a handful of testable, potentially useful hits that we could go through systematically and see which ones emerged with desirable properties," says Gradinaru.

Through this selection process, the researchers identified a variant dubbed AAV-PHP.B as a top performer. They gave the virus its acronym in honor of the late Caltech biologist Paul H. Patterson because Deverman began this work in Patterson's group. "Paul had a commitment to understanding brain disorders, and he saw the value in pushing tool development," says Gradinaru, who also worked in Patterson's lab as an undergraduate student.

To test AAV-PHP.B, the researchers used it to deliver a gene that codes for a protein that glows green, making it easy to visualize which cells were expressing it. They injected the AAV-PHP.B or AAV9 (as a control) into different adult mice and after three weeks used the amount of green fluorescence to assess the efficacy with which the viruses entered the brain, the spinal cord, and the retina.

"We could see that AAV-PHP.B was expressed throughout the adult central nervous system with high efficiency in most cell types," says Gradinaru. Indeed, compared to AAV9, AAV-PHP.B delivers genes to the brain and spinal cord at least 40 times more efficiently.  

"What provides most of AAV-PHP.B's benefit is its increased ability to get through the vasculature into the brain," says Deverman. "Once there, many AAVs, including AAV9 are quite good at delivering genes to neurons and glia."

Gradinaru notes that since AAV-PHP.B is delivered through the bloodstream, it reaches other parts of the body. "Although in this study we were focused on the brain, we were also able to use whole-body tissue clearing to look at its biodistribution throughout the body," she says.

Whole-body tissue clearing by PARS CLARITY, a technique developed previously in the Gradinaru lab to make normally opaque mammalian tissues transparent, allows organs to be examined without the laborious task of making thin slide-mounted sections. Thus, tissue clearing allows researchers to more quickly screen the viral vectors for those that best target the cells and organs of interest.

"In this case, the priority was to express the gene in the brain, but we can see by using whole-body clearing that you can actually have expression in many other organs and even in the peripheral nerves," explains Gradinaru. "By making tissues transparent and looking through them, we can obtain more information about these viruses and identify targets that we might overlook otherwise."

The biologists conducted follow-up studies up to a year after the initial injections and found that the protein continued to be expressed efficiently. Such long-term expression is important for gene therapy studies in humans. 

In collaboration with colleagues from Stanford University, Deverman and Gradinaru also showed that AAV-PHP.B is better than AAV9 at delivering genes to human neurons and glia.

The researchers hope to begin testing AAV-PHP.B's ability to deliver potentially therapeutic genes in disease models. They are also working to further evolve the virus to make even better performing variants and to produce variants that target certain cell types with more specificity.

Deverman says that the CREATE system could indeed be applied to develop AAVs capable of delivering genes specifically to many different cell types. "There are hundreds of different Cre transgenic lines available," he says. "Researchers have put Cre recombinase under the control of gene regulatory elements so that it is only made in certain cell types. That means that regardless of whether your objective is to target liver cells or a particular type of neuron, you can almost always find a mouse that has Cre recombinase expressed in those cells."

"The CREATE system gave us a good hit early on, but we are excited about the future potential of using this approach to generate viruses that have very good cell-type specificity in different organisms, especially the less genetically tractable ones," says Gradinaru. "This is just the first step. We can take these tools and concepts in many exciting directions to further enhance this work, and we—with the Beckman Institute and collaborators—are ready to pursue those possibilities." 

The Beckman Institute at Caltech recently opened a resource center called CLOVER (CLARITY, Optogenetics, and Vector Engineering Research Center) to support such research efforts involving tissue clearing and imaging, optogenetic studies, and custom gene-delivery vehicle development. Deverman is the center's director, and Gradinaru is the principal investigator.

Additional Caltech authors on the paper, "Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain," are Sripriya Ravindra Kumar, Ken Y. Chan, Abhik Banerjee, Wei-Li Wu, and Bin Yang, as well as former Caltech students Piers L. Pravdo and Bryan P. Simpson. Nina Huber and Sergiu P. Pasca of Stanford University School of Medicine are also coauthors. The work was supported by funding from the Hereditary Disease Foundation and the Caltech-City of Hope Biomedical Initiative, a National Institutes of Health (NIH) Director's New Innovator Award, the NIH's National Institute of Aging and National Institute of Mental Health, the Beckman Institute, and the Gordon and Betty Moore Foundation.

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Delivering Genes Across the Blood-Brain Barrier
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Caltech biologists have developed a vector capable of noninvasive delivery of genetic cargo throughout the adult central nervous system.

Rosens Recharge Support for Bioengineering

Caltech board chair emeritus and longtime Compaq chairman Benjamin M. (Ben) Rosen (BS '54) and his wife, Donna, have made a bequest commitment to advance scientific exploration at the intersection of biology and engineering. It is anticipated that the couple's latest gift may double the endowment for the Donna and Benjamin M. Rosen Bioengineering Center.

Established in 2008 with $18 million from the Benjamin M. Rosen Family Foundation of New York, the Rosen Center has become a hub for research and educational initiatives that bring together applied physics, chemical engineering, synthetic biology, computer science, and more.

"Just as we had the digital revolution in the last century, we are having a biological sciences revolution in this century," Ben Rosen says. "And Caltech is the place to be."

Read more on the Caltech giving site.

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Rosens Recharge Support for Bioengineering
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Caltech board chair emeritus Ben Rosen (BS ’54) and his wife Donna have made a commitment to scientific exploration at the intersection of biology and engineering.

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