Whenever we do anything—from breathe and run to stay healthy and live long lives—there are combinations of genes that had to do their job just right. But sometimes, a single faulty gene can lead to a major health problem.
One easy example is cystic fibrosis. We get one copy of the CFTR gene from our mom and one copy from our dad. As long as we have just one functional copy, we’re fine, and we might never know we inherited a faulty copy from the other parent. But if we inherit two faulty copies of the CFTR gene, we will develop cystic fibrosis. And this is why there are now drugs to treat cystic fibrosis at the genetic level.
UNC Health scientists at the UNC School of Medicine are attempting to do the same thing with Angelman syndrome, a severe neurodevelopmental disorder with no cure and limited treatments. Children born with this condition will develop severe intellectual and developmental disabilities, often endure seizures, and have problems with speech, balance, movement and sleep.
All of these symptoms arise because Angelman babies do not inherit a working copy of their moms’ UBE3A gene. Either the maternal version of the gene is nonexistent at birth, or the gene is mutated and does not lead to enough production of the UBE3A protein, or enzyme. Without this protein, our brain cells and nervous systems simply cannot function properly.
But what about the dad’s copy of the UBE3A gene? If that works, then wouldn’t kids be spared from developing the worst of Angelman syndrome?
That’s the big question UNC Health researchers led by Mark Zylka, PhD, want to answer. Dr. Zylka leads the UNC Neuroscience Center, and his lab has been working tirelessly to find out if dad’s copy of the gene could pick up the slack.
“Turns out, the paternal copy of UBE3A is typically silenced in our healthy neurons,” Dr. Zylka says. “So the loss of maternal UBE3A results in a complete absence of the UBE3A enzyme in most areas of the brain. That’s crucial because the job of UBE3A is to target proteins for degradation.”
What this means is inside us right now are enzymes with one job—to break down and dispose of molecules, such as proteins that we don’t need anymore. We need this degradation process to happen without a hitch to ensure our brain cells function properly.
When mom’s UBE3A doesn’t work, there still remains a long strand of RNA genetic code that blocks dad’s copy of the gene from producing the UBE3A enzyme.
Members of Dr. Zylka’s lab led by Justin Wolter, PhD, and Giulia Fragola,PhD, devised a way to cut out that troublesome RNA to free dad’s UBE3A. For that, they used gene-editing technology called CRISPR-Cas9.
CRISPR-Cas9 is DNA code that organisms use to find and destroy troublesome DNA of foreign invaders, such as bacteria. Scientists realized they could harness the natural power of CRISPR-Cas9 to manipulate genetic codes in more complex organisms.
Under Dr. Zylka’s guidance, Drs.. Wolter and Fragola determined how to use CRISPR-Cas9 to snip the genetic code in brain cell cultures grown in the lab. With promising preliminary findings in hand, Dr. Zylka received grants from the National Institutes of Health, the Angelman Syndrome Foundation, and the Simons Foundation to test his theory in human neurons and in a mouse model of the disease.
Dr. Wolter and Hanqian Mao, PhD, in the Zylka lab, and other UNC colleagues packaged their CRISPR-Cas9 technology inside an adeno-associated virus (AAV), which is a classic gene therapy delivery system.
Think of it like this: an AAV is like a common cold virus, notoriously good at getting into cells. If you strip out the viral genes that make you get a cold, then you can stuff it with a bit of genetic code to do something beneficial. In this sense, the AAV is just an Amazon Prime van delivering whatever you ordered. AAV technology is well understood and currently in experimental use for multiple sclerosis, eye disease, hemophilia, and other conditions. It is also being used in vaccines, including some for coronavirus disease 2019 (COVID-19).
So, Dr. Zylka’s lab packed their Amazon Prime AAV with fancy gene editing technology and sent it directly to human neurons in cell cultures and into animal models of Angelman syndrome—animals with deleted copies of maternal UBE3A.
The researchers found that embryonic and early post-birth treatment in mice reversed the same sort of major physical and behavioral characteristics found in human Angelman syndrome patients. Remarkably, a single injection of AAV woke the paternal UBE3A for at least 17 months in mice, and the research suggests that this beneficial effect is likely to be permanent. The researchers also showed that this approach was effective in human neurons in lab cell cultures.
Their work was published in Nature..
“We were blown away when we got these results,” Dr. Zylka says. “No other treatments currently being pursued for Angelman syndrome last this long, nor do they treat as many symptoms. I am confident others will eventually recognize the advantages of detecting the mutation that causes Angelman syndrome before birth and treating shortly thereafter.”
Dr. Wolter adds, “Since we learned we could reduce the severity of Angelman syndrome in mice, we are now focused on refining our approach in ways that will be suitable for use in humans.”
While working to translate this research into the clinic, Dr. Zylka’s lab will collaborate with researchers at the Carolina Institute for Developmental Disabilities (CIDD) to identify symptoms in babies that have the genetic mutation that causes Angelman syndrome.
Dr. Zylka’s lab is working with CIDD researchers led by CIDD director Joseph Piven, MD, to use brain imaging and behavior observations to identify symptoms associated with Angelman syndrome in infants. Anecdotal reports suggest these infants have difficulty feeding and reduced muscle tone, but these and other early symptoms have not been rigorously studied yet.
“The idea is to use genetic tests to identify babies that are likely to develop Angelman syndrome, treat prenatally or around the time of birth, and then use these early symptoms as ‘endpoints’ so we can evaluate how effective the treatment is in a clinical trial,” Dr. Zylka says. “We are incredibly excited to keep this work moving forward with the hope of helping children and families overcome this debilitating condition. Support from the NIH, the Simons Foundation, and the Angelman Syndrome Foundation was essential for moving this work forward.”
To support this research, go to the UNC Health Foundation, which leads funding efforts for UNC School of Medicine and UNC Health research efforts.
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