Abstract - 2018 - Research and Practice in Thrombosis and Haemostasis
Can CRISPR Fix A Childhood Brain Disorder?
When brain development gets off to a bad start, the consequences are lifelong.
One example is a condition called SCN2A haploinsufficiency, in which children are born with just one functioning copy of the SCN2A gene — instead of the normal two. They develop defects in the connections, or synapses, between some of their brain cells that affect how well they can send signals. As a result, they do not learn to speak, and many of them experience seizures.
Now, scientists at UC San Francisco have used a version of the gene-editing technology CRISPR to ameliorate some of these problems in mice, which can be engineered to carry the same mutation that humans do. But rather than trying to edit the defective copy of the gene, the scientists just turned up the volume on the healthy one.
The procedure worked in mice that were roughly equivalent in age to 10-year-old children, a clue that the brain may still be amenable to treatment well after much of its development has been completed. This is likely because SCN2A haploinsufficiency compromises how the brain fine-tunes its signals, but it does not affect other aspects of brain development.
The study, which was supported by the National Institutes of Health (NIH), appears in Nature on Sept. 17.
"We were surprised to see that the anatomy of the brain is totally intact — the synapses are there, but they fail to mature when there isn't enough SCN2A," said Kevin Bender, PhD, a professor in the UCSF Weill Institute for Neurosciences and co-senior author of the study. "By ramping up SCN2A levels in the brain, we brought those synapses online and restored signaling that prevented seizures."
Two copies of SCN2ANerve cells (pink) with two functioning copies of the SCN2A gene easily connect with other nerve cells and produce normal-sized, short wires (green).
One copy of SCN2ANerve cells with only one functioning copy of the SCN2A gene grew long wires that were unable to make good connections.
Restored levels of of SCN2AWhen treated with CRISPRa, these cells produced normal levels of the SCN2A protein, despite having only one functioning copy of the gene. These cells produced normal-length wires that could easily connect with other nerve cells.
Images by Tamura et al, Nature.
A lighter touch from CRISPRMore than a decade ago at UCSF, a new type of CRISPR was developed by Jonathan Weissman, PhD. It could find a gene and trigger its expression without creating DNA edits, and was dubbed CRISPRa, for "activation."
"This can compensate for the shortage of a gene that results from having just one good copy," said Nadav Ahituv, PhD, director of the UCSF Institute for Human Genetics, professor in the Department of Bioengineering and Therapeutic Sciences, and co-senior author of the paper.
In 2018, a team led by Ahituv used CRISPRa to treat a model of severe obesity in mice that was caused by the loss of one copy of an obesity-related gene.
Bender heard about Ahituv's success with CRISPRa and obesity, and realized it might also resolve the neurodevelopmental issues that come with insufficient SCN2A. The two UCSF scientists teamed up to see if it could work.
Too little SCN2A, too little learningSCN2A haploinsufficiency, which results in half the normal level of SCN2A, can lead to epilepsy, neurodevelopmental delay, and autism. Less SCN2A protein, in turn, means that brain cells cannot adjust their signals, like turning the dial of a radio to find the right station. Such adjustment is the basis for how the brain learns.
"This persistent function of SCN2A is critical for your ability to be human and experience and learn about the world," Bender said.
Making a model of diseaseLast year, Bender's team discovered a quirk in the eye movements of mice engineered to have SCN2A haploinsufficiency — something they later observed in children with the disorder. The quirk could serve as the basis for an easy clinical test for SCN2A haploinsufficiency in humans. The finding also meant they could study the disorder more readily in mice, since, according to Bender, it's rare for mutations that are found in people with autism to result in similar problems in mice. The Bender laboratory meticulously tested what this lack of SCN2A did to mice. Having less SCN2A made the mice prone to seizures and altered signals in their brain.
"Thanks to the beautiful characterization of physiology and behavior in the mice done by Kevin and his lab, we knew what needed to be restored to show that our CRISPRa approach was working," Ahituv said.
CRISPRa restores SCN2A levels and healthier brain functionA therapy like this in the clinic could improve their ability to talk and even live independently," Bender said. "We hope our work can help make these dreams a reality."
Kevin Bender, PhD
Ahituv's lab designed the CRISPRa to find the healthy copy of SCN2A and dial it up so it produced the same amount of protein that two copies of the gene would. And Bender's lab tested it in mice with SCN2A haploinsufficiency.
The CRISPRa intervention increased levels of SCN2A throughout the brain. The scientists saw normal amounts of SCN2A in nerve cells. But these animals had grown up with too little SCN2A from birth, and the mice were now a few weeks old, which is akin to a pre-adolescent human.
Would the additional protein be enough to treat the condition?
Remarkably, the additional SCN2A protein gave new life to the existing neural connections in these mice. Their brain signals looked normal, and they no longer were prone to seizures.
The intervention worked both when the CRISPRa was introduced directly to the brain and also when it was injected into the blood. Regel Therapeutics has licensed this technology from UCSF to treat patients with SCN2A haploinsufficiency disorders.
A key consideration will be how the brain responds to adding more SCN2A protein. The team was encouraged to find that it didn't harm healthy mice, those with two functioning copies of the gene. "Too much of any protein might cause a lot of trouble," Ahituv said. "We found that there is a natural limit to levels of this protein in SCN2A mice, but future therapies will need to confirm the safety of the approach in humans."
For such a severe disorder, the prospect of a treatment that works well into childhood would be miraculous.
"A therapy like this in the clinic could improve their ability to talk and even live independently," Bender said. "We hope our work can help make these dreams a reality."
Authors: Other UCSF authors are Serena Tamura, PhD, Andrew D. Nelson, PhD, Perry W.E. Spratt, PhD, Elizabeth C. Hamada, Xujia Zhou, PhD, Henry Kyoung, Zizheng Li, Coline Arnould, PhD, Vladyslav Barskyi, Beniamin Krupkin, MS, Kiana Young, Jingjing Zhao, PhD, Stephanie S. Holden, PhD, Atehsa Sahagun, Caroline M. Keeshen, PhD, Roy Ben-Shalom, PhD, Sunrae E. Taloma, Selin Schamiloglu, MS, Ying C. Li, MD, PhD, Jeanne T. Paz, PhD, Stephan J. Sanders, PhD, and Navneet Matharu, PhD. For all authors, see the paper.
Funding: This work was supported by the National Institutes of Health (P30 DK063720, S10 1S10OD021822-01, R01 MH125978: KJB; F32 MH125536 and K99 MH135209: ADN; R01 NS078118 and R01 NS121287: JTP; R01 MH115045, R01 NS 108874, and R01 MH118298: JQP; T32 GM007449: SSH); SFARI (629287, 513133), the Broad Institute Target Practice Initiative, the Autism 955 Science Foundation, the Weill Neurohub Investigator Program, the Natural Sciences and Engineering Research Council of Canada, the Ford Foundation Dissertation Fellowship, and the Weill Foundation.
Disclosures: Ahituv and Matharu are co-founders of Regel Therapeutics. Ahituv, Matharu, and Bender are scientific advisors to Regel Therapeutics. For all disclosures see the paper.
Disrupted Flow Of Brain Fluid May Underlie Neurodevelopmental Disorders
The brain floats in a sea of fluid that cushions it against injury, supplies it with nutrients and carries away waste. Disruptions to the normal ebb and flow of the fluid have been linked to neurological conditions including Alzheimer's disease and hydrocephalus, a disorder involving excess fluid around the brain.
Researchers at Washington University School of Medicine in St. Louis created a new technique for tracking circulation patterns of fluid through the brain and discovered, in rodents, that it flows to areas critical for normal brain development and function. Further, the scientists found that circulation appears abnormal in young rats with hydrocephalus, a condition associated with cognitive deficits in children.
The findings, available online in Nature Communications, suggest that the fluid that bathes the brain -- known as cerebrospinal fluid -- may play an underrecognized role in normal brain development and neurodevelopmental disorders.
"Disordered cerebrospinal fluid dynamics could be responsible for the changes in brain development we see in children with hydrocephalus and other developmental brain disorders," said senior author Jennifer Strahle, MD, an associate professor of neurosurgery, of pediatrics, and of orthopedic surgery. As a pediatric neurosurgeon, Strahle treats children with hydrocephalus at St. Louis Children's Hospital. "There's a whole host of neurologic disorders in young children, including hydrocephalus, that are associated with developmental delays. For many of these conditions we do not know the underlying cause for the developmental delays. It is possible that in some of these cases there may be altered function of the brain regions through which cerebrospinal fluid is circulating."
Much research has been conducted mapping the drainage of cerebrospinal fluid in the brains of adults. However, it is not well known how cerebrospinal fluid interacts with the brain itself. Cerebrospinal fluid pathways in the brain likely vary with age, as young children have not yet developed the mature drainage pathways of adults.
Strahle; first author Shelei Pan, an undergraduate student; and colleagues developed an X-ray imaging technique using gold nanoparticles that allowed them to visualize brain circulation patterns in microscopic detail. Using this method on young mice and rats, they showed that cerebrospinal fluid enters the brain through small channels primarily at the base of the brain, a route that has not been seen in adults. In addition, they found that cerebrospinal fluid flows to specific functional areas of the brain.
"These functional areas contain specific collections of cells, many of which are neurons, and they are associated with major anatomic structures in the brain that are still developing," Strahle said. "Our next steps are to understand why cerebrospinal fluid is flowing to these neurons specifically and what molecules are being carried in the cerebrospinal fluid to those areas. There are growth factors within the cerebrospinal fluid that may be interacting with these specific neuronal populations to mediate development, and the interruption of those interactions could result in different disease pathways."
Further experiments showed that hydrocephalus reduces cerebrospinal fluid flow to distinct neuron clusters. Strahle and colleagues studied a form of hydrocephalus that affects some premature infants. Babies born prematurely are vulnerable to brain bleeding around the time of birth, which can lead to hydrocephalus and developmental delays. Strahle and colleagues induced a process in young rats that mimicked the process in premature babies. After three days, the tiny channels that carry cerebrospinal fluid from the outer surface of the brain into the middle were fewer and shorter, and circulation to 15 of the 24 neuron clusters was significantly reduced.
"The idea that cerebrospinal fluid can regulate neuronal function and brain development isn't well explored," Strahle said. "In the setting of hydrocephalus, it's common to see cognitive dysfunction that persists even after we successfully drain the excess fluid. The disordered cerebrospinal fluid dynamics to these functional regions of the brain may ultimately affect brain development, and normalizing flow to these areas is a potential approach to reducing developmental problems. It is an exciting field, and we are only at the beginning of understanding the diverse functions of cerebrospinal fluid."
Cerebral Palsy And Sleep Disorders
Cerebral palsy (CP) is a complex neurodevelopmental condition stemming from early brain injury that affects movement, posture, and motor skills. Frequently, children with CP are at an increased risk of developing sleep disorders, which can encompass difficulties in initiating and maintaining sleep, sleep disordered breathing, excessive daytime sleepiness, and a range of parasomnias. These sleep disturbances are not only a source of additional morbidity but may also exacerbate core issues such as cognitive impairment, behavioural challenges, and reduced quality of life. Research in this area seeks to clarify the prevalence and underlying mechanisms of sleep disturbances in CP, and to develop robust, multidisciplinary management strategies that address both neurological and sleep-specific factors. The global significance of understanding sleep in CP lies in its potential to inform personalised treatment plans and improve overall outcomes for affected children and their families.
Research from Nature PortfolioNo recent Nature Portfolio content available
Research from all publishersRecent studies have underlined the importance of employing objective measures to assess sleep disturbances in children with CP. For instance, recent work using actigraphy has demonstrated significant concordance with traditional caregiver-reported sleep protocols, reinforcing the role of technology in enhancing sleep diagnostics [1]. In parallel, several investigations have focused on the behavioural and neurological correlates of disrupted sleep in this population. Research has flagged notable associations between sleep disturbances and the severity of CP symptoms, including motor impairment and comorbid epilepsy, suggesting that sleep disorders in CP are multifactorial in origin [2]. Systematic reviews and meta-analyses have further highlighted that children with CP exhibit a higher prevalence of sleep problems compared with typically developing peers, underscoring the need for routine screening and targeted interventions [3]. Moreover, studies analysing sleep-related characteristics in various CP subgroups have provided evidence that age and pain are significant predictors of abnormal sleep patterns, thus paving the way for interventions that can be tailored to the severity of CP and individual pain profiles [4]. Collectively, these contemporary findings stress the importance of integrative assessment methods that combine objective monitoring with caregiver insights, thereby improving both diagnosis and management of sleep disorders in children with cerebral palsy.
Technical TermsCerebral Palsy: A group of permanent movement and posture disorders caused by non-progressive disturbances in the developing infant brain.
Sleep Disorders: Conditions that affect the ability to sleep well on a regular basis, including problems with sleep initiation, maintenance, breathing, or quality that result in distress or impaired daytime functioning.
Actigraphy: A non-invasive methodology using wearable devices to monitor and record movement, thereby providing an objective measure of the sleep-wake cycle.
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