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In a breakthrough, researchers have utilized CRISPR-Cas9 gene editing in mice with a form of Fragile X Syndrome to alter gene expression, resulting in reduction of repetitive behaviors. Fragile X Syndrome (FXS) is the most commonly identified single-gene cause of autism spectrum disorder (ASD).
CRISPR, (Clustered Regularly Interspaced Short Palindromic Repeats), is a technology that allows scientists to edit genes in living cells. It holds an array of applications, not only from curing genetic diseases, detecting and treating cancers, treating HIV by blocking replication in human cells, but also the incredible feat of editing DNA in live human embryos.
The investigators in this study used gold nanoparticles to deliver the DNA-cutting Cas9 enzyme into the brain - a technique developed at the UC Berkeley, and referred to as CRISPR-Gold. Using this approach, the researchers were able to edit the gene for a neurotransmitter receptor and reduce the repetitive behavior characteristic of fragile X syndrome (FXS).
The significance of this feat—demonstrating how genetic editing resulted in a change in behavior—is that it may potentially open the door for treating other forms of autism that also have genetic origins. Since exaggerated repetitive behaviors are common aspects of behavior observed in autism spectrum disorders, reduction of these behaviors could have life changing consequences for affected children and their parents.
Results of the study were published online today in the journal, Nature Biomedical Engineering.
"There are no treatments or cures for autism yet, and many of the clinical trials of small-molecule treatments targeting proteins that cause autism have failed," said study leader Hye Young Lee, assistant professor of cellular and integrative physiology at the University of Texas Health Science Center at San Antonio in a press release. "This is the first case where we were able to edit a causal gene for autism in the brain and show rescue of the behavioral symptoms."
The big advantage of using CRISPR-Gold is that it does not rely upon using viruses to get Cas9 into the body --something that traditional CRISPR has utilized.
"The really compelling thing about this paper is that Hye Young was able to show that if you injected CRISPR-Gold into the brain, you could knock out disease-causing genes and actually see fairly significant behavioral changes," said CRISPR-Gold inventor Niren Murthy, a UC Berkeley professor of bioengineering. "This is the first time anyone had ever shown that with non-viral delivery."
Individuals with autism spectrum disorders have difficulty interacting with other people , along with exaggerated repetitive behaviors, such as hand-flapping and rocking back and forth.
Since ASD likely has multiple etiologies—including a number of genetic mutations, studying single-gene disorders like FXS are a much easier way of evaluating the causes along with possible treatments. ASD is known to affect slight more than 1% of all children; however, FXS is also rare, occurring in one of every 4,000 boys and 6,000 girls.
This study is the first proof-of-concept study to show that the Cas9 protein can be shuttled into the brain to “knock out” a specific gene, which translates to therapeutic effects by noting an alteration in gene expression (less repetitive movements).
"If you inject CRISPR DNA using a virus, you can't control how much Cas9 protein and guide RNA are expressed, so injecting it via a virus has a potential problem," Lee said. "I think the CRISPR-Gold method is very cool because we can control the amount we wish to inject and that probably minimizes the side effects of using CRISPR, for example off-target effects."
The possibilities of treating other complex conditions-- opioid addiction, neuropathic pain, schizophrenia and epileptic seizures—seem more likely with the advantages of this advanced CRISPR-Gold technique, Murthy believes.
DownRegulating an Overstimulated Brain
For their breakthrough experiment--using mice with FXS--the investigators injected CRISPR-Gold, which carried the Cas9 complex into an area of the brain known as the striatum, an area that controls formation of habits or behaviors, including that related to repetitive behaviors common with ASD.
The Cas9 complex targeted an excitatory receptor, known as the metabotropic glutamate receptor 5 (mGluR5) , that is integral in communication between neurons (nerve cells) and is known to have abnormal function in FXS. The researchers were able to disable the gene for mGluR5, allowing them to decrease the exaggerated signaling between cells, thereby achieving a reduction in repetitive behaviors.
"Before this experiment, we didn't know if the mGluR5 receptor in the striatum was specifically involved in exaggerated repetitive behavior; that is an important biological finding of our study," Lee said.
Repetitive behaviors in FXS mice included repeated leaping and jumping as well as obsessive digging. CRISPR-Gold editing reduced digging behavior by nearly 30%, while the leaping went down by 70%. CRISPR-Gold reduced the number of mGluR5 genes in the striatum by 50%, which subsequently led to a 50% reduction in the number of receptor proteins.
In the past, pharmaceutical companies tried to inject small-molecule drugs into the blood system to block the same receptor--resulting in small reductions in repetitive behaviors- but mice became tolerant, and did not respond to subsequent treatments.
The CRISPR-Gold system, developed by Murthy, uses gold nanoparticles covered by a blanket of DNA chains that hold the Cas9 molecules, which are a combination of a gene-cutting enzyme and a guide RNA that searches for the mGluR5 gene. The package is enveloped in a polymer that helps it gain entry into the specifically-targeted cells.
Perspective on the Significance of CRISPR-Gold
"The findings of this study are groundbreaking for two reasons," offered Mitchell Ng, Principal and Managing Director of biotech investment firm Thessalus LLC. "First and foremost, the study shows CRISPR therapy leading to a tangible improvement in yet another previously incurable and terminal disease, Fragile-X autism."
Ng explained that "for years pharma companies such as Pfizer and Merck poured billions into small molecule treatments for Autism, Alzheimer's, Parkinson's, and other neurological diseases to no avail. Here's a study showing unprecedented improvement--but this is no surprise, given over the past year we've already seen CRISPR successfully treating other terminal diseases like Duchenne Muscular Dystrophy in clinical trials run by Sarepta Therapeutics."
"Second, and perhaps far more important," Ng explains "is the validation of CRISPR Gold as a superior delivery method. The CRISPR technology is proven to work but one of the main concerns among investors regarding potential commercialization has been delivery."
While the adenovirus vectors used to deliver CRISPR were effective in most cases, the main concern were issues including unwanted replication and cutting of random genes by the Cas9 enzyme.
"The CRISPR Gold nanoparticles are now shown to be superior in that they deliver the genetic package to the target and that's it, no unwanted replication, emphasized Ng. " Unlike before, there is a clear ability to control dosing, a huge implication in the future clinical and financial potential for the CRISPR technology."
"It is hard to overstate the importance of this: Over the past 11 months, three largest publicly traded CRISPR companies on the NASDAQ, companies we've all invested in, Editas Medicine, Crispr therapeutics, Intellia, each tripled in stock price and achieved billion-dollar market capitalizations. This news only points to their continued success and growth potential."
Dr. Kenneth Ng, co-managing director of Thessalus LLC, echoes these sentiments, concurring that "the most significant part of this study by far is in the proof of concept of CRISPR Gold. What Murthy's lab has done is significantly move forward the timeline and day by which CRISPR becomes not just a research tool but a widely commercialized and utilized clinical therapy."
"Unlike viral vectors, which like any viral infection are unpredictable and can disrupt undiseased genetic code, the Gold nanoparticles attached to the RNA target allow precise targeting and dosing, disappearing after use. This is a big leap over what existed before," added Ng.
Murthy's Accomplishments in Gene Editing
Murthy’s accomplishments are quite impressive: in 2017, he and his associates demonstrated that CRISPR-Gold could transport Cas9 into muscle cells, replacing a mutated gene with a normal gene to improve strength in mice with Duchenne muscular dystrophy.
He has taken this this one step further, demonstrating in this present study that CRISPR-Gold can successfully shuttle Cas9 into multiple types of different cells in the brain.
"We showed in this paper that we were also able to edit non-neurons: the microglia, which are part of the brain's immune system, and astrocytes, which support the neurons," Murthy said. "We actually edited those more efficiently than neurons, and those can play a very important role in a lot of diseases."
GenEdit, spun off from Murthy’s lab in 2016, plans to develop CRISPR-Gold therapies for a variety of genetic diseases. Dr. Kunwoo Lee, a PhD researcher in Murthy’s lab, and now serving as GenEdit’s CEO, launched the company in 2017 out of CITRIS Foundry, an accelerator at UC Berkley. Dr. George Church of Harvard and Matthew Rabinowitz, CEO of Natera are two of their current advisors.
"CRISPR-Gold can be used to treat a variety of genetic diseases, such as Huntington's disease," Lee said. "But it's not limited to monogenic diseases; it can also be used against polygenic diseases, once we figure out the entire network of genes involved."
What’s so important about this study, according to Murthy, is that GenEdit figured out the logistics to safely ship the CRISPR-Gold particles a long distance--from UC Berkeley to San Antonio--and reproduce them. This helped to remove an important issue that has limited the adoption of many other other nanotechnologies.
One fascinating project their team is now working on involves the development of CRISPR-Gold particles that can be injected directly into the central nervous system (CNS) via the spinal cord, obviating the requirement for brain surgery (craniotomy) to gain access to specific areas of the brain. Dr. Hye Young Lee believes that this might be as effective as directly injecting them into the striatum to reduce repetitive behavior. The hope is that this therapy will affect phenotypic expression of genes for social interaction associated with ASD.
