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CRISPR Gene Editing of Neurons in Prader-Willi Syndrome

I’m happy to see that fears about using CRISPR to edit human genes have dampened over the past year, but it’s still fun perusing the hyperbolic headlines:

They’re going to CRISPR people. What could possibly go wrong?”

“The Very Real Dangers of New Gene-Editing Technology”

“How Gene Editing Could Ruin Human Evolution”

(Ernesto del Aguiila, NHGRI)

Since being fortunate enough a few years ago to find myself one of only two journalists in the press room at a genetics conference with two of CRISPR/Cas9’s inventors, I’ve made an effort to highlight exciting, beneficial uses of gene editing techniques. DNA Science has looked at research applications of the technology in Huntington’s disease, sickle cell disease, and split-hand/foot malformation.

This week, another compelling study using CRISPR to interrogate a gene behind a disease appears in Human Molecular Genetics, from postdoctoral researcher Maeva Langouet, professor of genetics and genome sciences Marc Lalande, and their colleagues at the University of Connecticut. The condition is rare, devastating, and has an unusual origin.

“Failure To Thrive” Leads to Obesity

The first signs of Prader Willi Syndrome (PWS) aren’t especially distinctive or specific – a small infant with poor muscle tone is too weak to eat enough, leading to “failure to thrive.” By age 3, the child becomes better able to move around, and starts to gain weight – but keeps gaining. As metabolism slows, the thinness of infancy and toddlerhood paradoxically becomes obesity. When the child becomes obsessed with seeking food, reflecting damage to the brain’s seat of satiety in the hypothalamus, the diagnostic odyssey may begin to focus in on Prader-Willi syndrome.

Jayden at age 5, before the obsessive eating behavior began. Today at age 8, a locked pantry and refrigerator help him to maintain a normal weight.

The overeating worsens. No pill, therapy, or surgery can cure the condition, although interventions address specific symptoms, and control of diet and exercise are crucial. But parents have to take measures that may seem extreme to those not in their shoes – they lock refrigerators, kitchen cabinets, and garbage pails to keep their children from consuming so much that their stomachs burst. Controlling the environment counters the inability to control the behavior.

PWS is the most common genetic cause of life-threatening obesity in children, but it has other symptoms. Young people with the condition may have poor growth, sleep apnea, intellectual and/or learning disabilities, sex hormone deficiency, and exhibit odd repetitive behaviors, such as skin picking.

Yet parents can channel some behaviors into positive actions. That’s the case for Jake Vasiloff, whose story appears in The Columbus Dispatch. Banging on pots and pans as a toddler, and then a toy bongo drum to get him to go to doctor appointments, led to today’s 18-year-old drummer. Music and art are terrific medicines for many children with rare diseases.

Altered Epigenetics

PWS arises from an intriguing short stretch of chromosome 15 that is subject to a phenomenon called genomic imprinting – the gender of the parent who contributes the glitch to the fertilized ovum (and therefore the child) is important.

Normally, methyl (CH3) groups cover the region of the chromosome that comes from the mother, effectively silencing it so that RNA isn’t transcribed and protein not translated. The individual is ok as long as the corresponding paternal part of chromosome 15 is undisturbed. But if it’s missing, PWS results.

In 70% of individuals with PWS, about 5,000 DNA bases are deleted. Another 30% inherit both chromosome 15 sections from the mother (called uniparental disomy), and a small percentage of cases inherit PWS another way, from a mutation in an imprinting gene. About 1 in 15,000 newborns has PWS.

Interestingly, a nearby gene on chromosome 15 has the reverse imprinting pattern: the paternal copy is silenced and the maternal one works. If the maternal gene is missing, the child develops a different condition, Angelman syndrome. It causes autism and intellectual disability, an extended tongue, large jaw, poor muscle coordination, and characteristic arm flapping.

Lifting the Imprint

Theoretically, turning on the silenced maternal gene could counter PWS. In late 2016, researchers from Duke University announced using that approach against a protein called G9a with promising results in mice.

Now the University of Connecticut team has silenced the maternal part of chromosome 15 a different way, in neurons from patients.

They worked with induced pluripotent stem (iPS) cells that were derived from skin cells of PWS patients. Adding a specific cocktail of biochemicals coaxed the stem cells to divide and give rise to daughter cells that specialized as neural progenitor cells, which in turn gave rise to brain neurons. The researchers zeroed in on a specific gene that encodes a protein called ZNF274, which normally tethers the silencing machinery to the imprinted portion of the maternal chromosome 15. They deployed CRISPR/Cas9 to silence the ZNF274 gene so that there’d be no ZNF274 protein to shut off the maternal DNA.

It worked. The treated nerve cells turned on the maternal copy of the Prader-Willi region of chromosome 15. So could ZNF274 protein be a drug target? Alas its functions are too “promiscuous,” binding to different sites in the genome, to reign in potential off-target effects. But researchers now know the next steps – restrict the protein’s action to the part of the chromosome directly implicated in PWS.

For a disease without direct, disease-modifying treatment, a molecular correction is a promising beginning.

(Ironically, ZNF274 is a zinc finger protein, a “motif” found in nearly a tenth of our proteins that binds zinc atoms and helps control gene expression. Enzymes that cut certain zinc fingers – the zinc finger nucleases – have been used experimentally to selectively silence genes since 2009, and are a forerunner to CRISPR. I wrote about zinc finger nucleases to treat hemophilia here.)

The Foundation for Prader-Willi Research, the Cascade Fellowship, and the CT Regenerative Medicine Fund supported the new work. Thanks to the Foundation for providing the photo of Jayden.

 

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