Better ‘Mini Brains’ Could Help Scientists Identify Treatments for Zika-Related Brain Damage

UCLA researchers develop improved technique for creating brain tissue from stem cells

UCLA researchers have developed an improved technique for creating simplified human brain tissue from stem cells. Because these so-called “mini brain organoids” mimic human brains in how they grow and develop, they’re vital to studying complex neurological diseases.

In a study published in the journal Cell Reports, the researchers used the organoids to better understand how Zika infects and damages fetal brain tissue, which enabled them to identify drugs that could prevent the virus’s damaging effects.

The research, led by senior author Ben Novitch, could lead to new ways to study human neurological and neurodevelopmental disorders, such as epilepsy, autism and schizophrenia.

“Diseases that affect the brain and nervous system are among the most debilitating medical conditions,” said Novitch, UCLA’s Ethel Scheibel Professor of Neurobiology and a member of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA. “Mini brain organoids provide us with opportunities to examine features of the human brain that are not present in other models, and we anticipate that their similarity to the real human brain will enable us to test how various drugs impact abnormal or diseased brain tissue in far greater detail.”

For about five years, scientists have been using human pluripotent stem cells, which can create any cell type in the body, to develop mini brain organoids. But the organoids they produced have generally been difficult to use for research because they had highly variable structures and inconsistent cellular composition, and because they didn’t correctly mimic the layered structure of the brain and were too small — often no bigger than the head of a pin. They also didn’t survive very long in the laboratory and contained neural tissue that was difficult to classify in relation to real human brain tissue.

The organoids developed by Novitch’s group have a stratified structure that accurately mimics the human brain’s onion-like layers, they survive longer and have a larger and more uniform shape.

To create the brain organoids, Novitch and his team made several modifications to the methods that other scientists used previously: The UCLA investigators used a specific number of stem cells and specialized petri dishes with a modified chemical environment; previous methods used varying amounts of cells and a different type of dish. And they added a growth factor called LIF, which stimulated a cell-signaling pathway that is critical for human brain growth.

The researchers found critical similarities between the organoids they developed and real human brain tissue. Among them: The organoids’ anatomy closely resembled that of the human cortex, the region of the brain associated with thought, speech and decision making; and a diverse array of neural cell types commonly found in the cortex were all present in the organoids, and they exhibited electrical activities and network function, meaning they were capable of communicating with one another much like the neural networks in the human brain do.

The UCLA scientists also found that they could modify their methodology to make other parts of the brain including the basal ganglia, which are involved in the control of movement and are affected by neurodegenerative conditions such as Parkinson’s disease and Huntington’s disease.

“While our organoids are in no way close to being fully functional human brains, they mimic the human brain structure much more consistently than other models,” said Momoko Watanabe, a UCLA postdoctoral fellow and the study’s first author. “Other scientists can use our methods to improve brain research because the data will be more accurate and consistent from experiment to experiment and more comparable to the real human brain.”

When the team exposed the organoids to Zika, they discovered specifically how the virus destroys neural stem cells, the cells from which the brain grows during fetal development. Novitch’s team found that there are four specific molecules, called receptors, on the outer surface of neural stem cells; previous studies have indicated that the Zika virus could bind to these receptors and infect the cells. The researchers then mapped the changes that occur in the neural stem cells after Zika infection, presenting a clearer picture of how the virus infiltrates and harms fetal brain tissue.

Zika is associated with an unusually high incidence of fetal brain damage, so understanding how neural stem cells are affected by the virus could be an important new step toward a treatment.

The researchers tested several drugs on the Zika-infected organoids. They found three that are effective at blocking the virus’s entry into the brain tissue, including two that protected neural stem cells by preventing the interaction between the virus and entry receptors on the neural stem cells. In previous studies by Novitch and other UCLA colleagues, one of those drugs reduced brain damage in fetal mice infected with Zika.

“Many neurological diseases or conditions arise from defects in the way one neuron communicates with another or from the way an external factor, such as a virus, interacts with neural cells,” Novitch said. “If we can focus in at the level of cellular communication, we should be able to model those undesirable cellular interactions and counteract them with drugs or other therapies.”

The team plans to continue using its improved organoids to better understand human brain development and to learn more about autism spectrum disorders, epilepsy and other neurological conditions.

The experimental drugs used in the preclinical study have not been tested in humans or approved by the Food and Drug Administration for treating Zika in humans.

Zika Virus Protein Mapped to Speed Search for Cure

A recently-published study shows how Indiana University scientists are speeding the path to new treatments for the Zika virus, an infectious disease linked to birth defects in infants in South and Central America and the United States.

Cheng Kao, a professor in the IU Bloomington College of Arts and Sciences‘ Department of Molecular and Cellular Biochemistry, has mapped a key protein that causes the virus to reproduce and spread.

“Mapping this protein provides us the ability to reproduce a key part of the Zika virus in a lab,” Kao said. “This means we can quickly analyze existing drugs and other compounds that can disrupt the spread of the virus. Drugs to target the Zika virus will almost certainly involve this protein.”

The World Health Organization reports that more than 1 million people in 52 countries and territories in the Americas have been infected with the Zika virus since 2015. The disease has also been confirmed to cause microcephaly in more than 2,700 infants born to women infected with the virus while pregnant. Symptoms include neurological disorders and a head that is significantly smaller than normal.

The virus is also transmissible through sexual activity and can trigger an autoimmune disease in adults called Guillain-Barre syndrome.

The IU-led study, conducted in collaboration with Texas A&M University, revealed the structure of the Zika virus protein NS5, which contains two enzymes needed for the virus to replicate and spread. The first enzyme reduces the body’s ability to mount an immune response against infection. The other enzyme helps “kick off” the replication process.

“We need to do everything we can to find effective drugs against the Zika virus, as changes in travel and climate have caused more tropical diseases to move into new parts of the globe,” said Kao, who has also spent 15 years studying the virus that causes hepatitis C.

“We’ve learned a lot of lessons about how to fight this class of virus through previous work on hepatitis C, as well as other work on the HIV/AIDS virus,” he added.

In addition, Kao said, the study showed that the Zika virus protein is similar in structure to proteins from viruses that cause dengue fever, West Nile virus, Japanese encephalitis virus and hepatitis C, which prompted the team to test several compounds that combat those diseases. The team also tested other compounds to disrupt the virus’s replication.

“Drugs approved to treat hepatitis C and compounds in development to treat other viral diseases are prime candidates to use against the Zika virus,” Kao said. “We’re continuing to work with industry partners to screen compounds for effectiveness against the NS5 protein.”

Other IU Bloomington authors on the study were Guanghui Yi and Yin-Chih Chuang in the Department of Molecular and Cellular Biochemistry and Robert C. Vaughan in the Department of Biology. Additional authors were Baoyu Zhao and Pingwei Li of Texas A&M University and Banumathi Sankaran at Lawrence Berkeley National Laboratory.

The method used to reproduce the virus protein in the lab is the subject of a U.S. patent application filed by the IU Research and Technology Corp.

The study appears in the journal Nature Communications. It was supported in part by the Johnson Center for Innovation and Translational Research at IU Bloomington.

Gene Therapy Company Acquires Next Generation AAV Gene Therapy Vector Platform from The University of North Carolina at Chapel Hill

The University of North Carolina at Chapel Hill has licensed the exclusive worldwide rights of a next generation gene therapy AAV capsid portfolio to Abeona Therapeutics Inc., a clinical-stage biopharmaceutical company focused on developing therapies for life-threatening rare genetic diseases. The AIM vector system is a next generation platform of AAV capsids capable of widespread central nervous system gene transfer and can be used to confer high transduction efficiency for various therapeutic indications.

Studies indicate that AIM vectors can efficiently and broadly target CNS tissue, and may provide a treatment for patients that have inhibitory antibodies to natural AAV serotypes. Importantly, the AIM vector system may provide second-generation treatment approaches for patients that have received a previous AAV injection.

“As we continue to build out our orphan and rare disease drug portfolio and move additional programs into the clinic, this agreement with UNC continues the execution of our strategy to combine our expertise in advancing gene therapy programs with the development of a next-generation proprietary AAV vector platform,” stated Steven H. Rouhandeh, Executive Chairman. “We look forward to harnessing the clinical utility and therapeutic potential of the AIM vector system technology platform to address a broad range of rare genetic diseases.”

In addition to the AAV capsid library, the license also adds ABO-202, an AAV-based CLN1 program, to Abeona’s Batten pipeline.  ABO-202, developed at UNC by Steven Gray, Ph.D. with the support of The Saoirse Foundation, Taylor’s Tale, Hayden’s Batten Disease Foundation, and the Batten Disease Support and Research Association, is anticipated to enter clinical trials in 2017 for patients with infantile neuronal ceroid lipofuscinosis (INCL, infantile Batten disease), an inherited fatal genetic disease that primarily affects the nervous system.

Infantile neuronal ceroid lipofuscinosis (INCL) CLN1, also known as PPT1, encodes an enzyme called palmitoyl-protein thioesterase 1 that is insufficiently active in Infantile NCL. Infantile NCL (INCL or Santavuori-Haltia disease) begins between about ages 6 months and 2 years and progresses rapidly. Affected children fail to thrive and have microcephaly. Also typical are short, sharp muscle contractions called myoclonic jerks. These children usually do not reach age 5.

“ABO-202 has shown promising preclinical efficacy in INCL mice after delivery of a functioning copy of the CLN1 gene to cells of the central nervous system, by extending survival and preserving strength when administered early in the disease course,” noted Steven J. Gray, Ph. D, Assistant Professor, Department of Ophthalmology, Gene Therapy Center, University of North Carolina at Chapel Hill. “Our work in developing these novel, next generation AAV gene therapy vectors have the potential to further advance the field of AAV-based technologies by efficiently and specifically targeting the CNS, with a likelihood of avoiding antibodies endogenously generated by natural AAV serotypes.”

“The AIM vector system is a next generation AAV-based gene therapy technology platform that represents a transformational opportunity for Abeona. The AIM platform will allow us to leverage our current pipeline into second generation products for CNS and other tissue-specific delivery, and help provide an answer for patients that have existing inhibitory antibodies,” stated Timothy J. Miller, Ph.D., President & CEO. “In addition, we add another AAV-based product ABO-202 (AAV-CLN1) for treatment of patients with infantile neuronal ceroid lipofuscinosis (INCL), which builds on our expertise in developing treatments for patients with forms of Batten disease.”

Sophisticated ‘Mini-Brains’ Add to Evidence of Zika’s Toll on Fetal Cortex

Studying a new type of pinhead-size, lab-grown brain made with technology first suggested by three high school students, Johns Hopkins researchers have confirmed a key way in which Zika virus causes microcephaly and other damage in fetal brains: by infecting specialized stem cells that build its outer layer, the cortex.

The lab-grown mini-brains, which researchers say are truer to life and more cost-effective than similar research models, came about thanks to the son of two Johns Hopkins scientists and two other high school students who were doing summer research internships. They had the idea to make the equipment for growing the mini-brains with a 3-D printer. These so-called bioreactors, and the mini-brains they foster, should open other new and valuable windows into human brain development, brain disorders and drug testing — and perhaps even produce neurons for treatment of Parkinson’s disease and other disorders, the investigators say.

A report on the research appears online April 22 in the journal Cell.

“We have been working for three years to develop a better research model of brain development, and it’s fortunate we can now use this one to shed light on the major public health crisis posed by Zika infections,” says Hongjun Song, Ph.D., professor of neurology and neuroscience at the Johns Hopkins University School of Medicine’s Institute for Cell Engineering. “This more realistic, 3-D model confirms what we suspected based on what we saw in a two-dimensional cell culture: that Zika causes microcephaly — abnormally small brains and heads — mainly by attacking the neural progenitor cells that build the brain and turning them into virus factories.”

In recent years, researchers in various fields have begun to grow tiny organs from human stem cells to get a better view of development and disease, and speed the search for new drugs. But existing techniques for creating and working with mini-brains were limiting because of the organ’s complexity, Song says. Though the mini-brains themselves are about the size of a pinhead, the bioreactors where they grew were comparatively large, about the size of a soda can. That made working with the mini-brains expensive, given the high cost of the nutrients needed to cultivate human stem cells in the lab, he says, as well as the expense of chemical growth factors that guide the tissue to organize itself like a real brain. Few labs could afford to grow enough mini-brains to be useful for research, Song says, and those that did produced tissues with cells specialized for different parts of the brain mixed together at random.

Song and his wife and research partner, Guo-li Ming, M.D., Ph.D., professor of neurology, neuroscience, and psychiatry and behavioral sciences, found a way to improve the bioreactors from an unexpected source: their son and two other high school students, from New York and Texas, who spent a summer working in the lab. The students had worked with 3-D printers and thought they could be the key to producing a better bioreactor, one that would fit over commonly used 12-well laboratory plates and spin the liquid and cells inside at just the right speed to allow the cells to form brains.

Of course, it wasn’t that simple, Song says. Graduate student Xuyu Qian and postdoctoral fellow Ha Nam Nguyen, Ph.D., spent years determining factors such as what that optimum speed was, as well as which chemicals and growth factors should be added at what times to yield the desired result.

The group has so far used the new bioreactor, dubbed SpinΩ, to make three types of mini-brains mimicking the front, middle and back of a human brain. They used the forebrain, the first mini-brain with the six layers of brain cell types found in the human cortex, for the current study on Zika.

“One thing the mini-brains allowed us to do was to model the effects of Zika virus exposure during different stages of pregnancy,” says Ming. “If infection occurred very early in development, the virus mostly infected the mini-brains’ neural progenitor cells, and the effects were very severe. After a while, the mini-brains would stop growing and disintegrate. At a later stage, mimicking the second trimester, Zika still preferentially infected neural progenitor cells, but it also affected some neurons. Growth was slower, and the cortex was thinner than in noninfected brains.”

These different effects correspond to what clinicians have seen in infants born to women who contracted Zika during pregnancy, as well as miscarriages, she notes, namely that the earlier in pregnancy Zika infection occurs, the more severe its effects.

The research group’s next step will be to test drugs already FDA-approved for other conditions on the mini-brains to see whether one might provide some protection against Zika. And they included 3-D printing files for SpinΩ in the new paper so that researchers anywhere can print their own bioreactors with just a few hundred dollars in materials. Song says one possible future use could be to grow so-called dopaminergic neurons for transplant, to replace those that die off in Parkinson’s disease. “This is the next frontier of stem cell biology,” he says.