Inside a Yale lab, Adele Ricciardi is pouring small amounts of a clear liquid into two glass vials. Dressed in a white lab coat and wearing pink-rimmed lab glasses, the M.D. and Ph.D. student at Yale is preparing PNA molecules, which are a type of synthetic DNA.
These molecules will ultimately be encapsulated in microscopic nanoparticles. These tiny particles, which are billionths of a meter in diameter, are designed to deliver the PNA molecules to specifically targeted cells where they can attach themselves to mutated DNA within living mice or human cells, and rewrite parts of their genetic code.
Ricciardi works in the lab of Peter Glazer, a Yale geneticist and M.D. Glazer’s lab is one of several in Connecticut on the frontier of one of biology’s most exciting emerging fields: gene editing, the process of correcting, deleting or inserting DNA into the genome of a living human or animal.
Gene-editing techniques, though they have become commonplace in labs, have not been widely used in humans. That could soon change, experts say. In the coming years there is hope that emerging gene-editing technologies will provide a host of treatments and possibly even cures for a wide range of conditions.
Many Connecticut researchers are on the cutting edge of this field. Here we take a closer look at some of the research currently being done in the state and some of the misconceptions surrounding gene editing.
Will gene editing lead to designer babies?
A major misconception about gene editing is a belief that it will soon lead to parents selecting preferred physical and mental traits for their unborn babies, creating so-called “designer babies” who are better-looking, faster, smarter humans. This is not the direction gene-editing technology is headed, experts say.
“A lot of that is a science-fiction type of viewpoint. That really has nothing to do with what people are working on now in gene editing,” Glazer says. “What my lab and most other academic labs, and some of the companies that have sprouted up, are trying to do is use gene editing in a very focused way to fix specific mutations in genes that are linked to very well-characterized diseases. Something like sickle cell anemia.
“If someone said, ‘Please make a human that can run fast,’ no one would know how to do that.”
Stormy Chamberlain, a geneticist at UConn Health, agrees. “People incorrectly assume that the only use for this technology is to make designer babies. This technology is going to be much more widely used and going to have a bigger impact on the development and testing of pharmaceuticals,” she says. “We all understand there’s a slippery slope between fixing a disease-causing gene and changing a trait or a characteristic that we find less desirable.”
Beyond the moral and ethical implications, there are also practical ones.
The gene-editing techniques that are currently being used on animals in labs are often imprecise and can have what are termed off-target effects, when changes occur to genes that are not being targeted by the therapy.
A far safer option for parents already exists through embryo testing and selection with in vitro fertilization. “If a couple knows that each of them are carriers for a [gene] variant that carries cystic fibrosis, what they might do is, rather than risk having a child with cystic fibrosis, they might undergo IVF and have each of the resulting embryos tested for whether or not they’ve inherited both mutations from the parents,” Chamberlain says. Assuming the disease is well understood and can be tested for in embryos, parents can then choose the embryos that are mutation free.
“If you can select the embryos that don’t have the disorder, there’s not a real reason to correct the gene-causing mutation,” she says, especially considering a gene-editing attempt might have an off-target effect.
What kind of genetic testing is being done with IVF?
Unlike gene editing of embryos, which does not occur in a clinical setting, genetic testing and embryo selection to avoid severe childhood disease and chronic conditions is fairly widespread.
Maurice Mahoney, an M.D. and professor emeritus of genetics and of obstetrics, gynecology and reproductive sciences at Yale School of Medicine, says this process is relatively common.
“The whole purpose is to allow selection for embryos without disease and reject for implantation embryos that have disease,” Mahoney says. “Some couples will extend that if they can to say, ‘We don’t even want our child to be a carrier of one of our mutations like each of us is.’ ”
Mahoney says that “selection on disease isn’t highly controversial overall in our society, but there are still ethical questions that are debated.
“[There are] people in the disability community who say, ‘You’re trying to identify us and say we’re not acceptable to join the human society.’ ” He adds that you have instances “where the parents want a child born with that disorder. The most talked-about communities where that practice occurs at times is in the deaf community and with people with short stature who want only children like themselves or like the people in the communities of people that they belong to.”
There is also debate about just how far this method should be taken. Currently it is used for childhood diseases, but there are those who would like to extend it and select embryos that likely have lower chances of cancer, heart problems or psychiatric disease such as schizophrenia or bipolar disease. Even as it becomes more plausible, Mahoney says “some people are very much opposed to selecting babies for the purpose of not having a baby born who has a higher risk for those type of disorders.”
Other questions arise when the technology is used to select embryos based on gender, not disease, which Mahoney says does occur sometimes. But he adds that most of the ideas associated with the term “designer babies” remain the stuff of imagination.
“Designing what your child is going to be like in terms of intellectual capacity, physical capacity, emotional capacity, those things which people have dreamed about, and wondered what you might do, we don’t know enough about it, and the ethical discourse about that is all over the place,” he says. “In general, mainstream prenatal diagnosis or IVF services aren’t talking about that and aren’t doing that.”
What gene-editing techniques are being studied in Connecticut?
At UConn Health, Chamberlain is exploring ways Angelman syndrome, an autism-like disorder, may one day be treatable with CRISPR-based gene-activating or -silencing treatments administered during a child’s first year of life. Angelman syndrome is a rare neuro-genetic disorder, with characteristics including developmental delay, lack of speech, seizures and walking and balance disorders. It is caused by a mutation of the UBE3A gene, and Chamberlain is developing strategies to fix that mutation using CRISPR-Cas9 to test therapeutic approaches in the lab. If it ultimately works, Chamberlain’s still-in-development method could have implications for autism treatments in the future.
CRISPR-Cas9 is the most common gene-editing tool. Consisting of RNA and an enzyme, and often described as a type of molecular scissors, CRISPR-Cas9 can cut DNA at specific sites and introduce new information that will be incorporated into the DNA as it repairs the cut. Modified versions of CRISPR-Cas9 can also turn off or on genes by binding to DNA and bringing with it proteins called activators and repressors, respectively.
“Autism is really a collection of distinct neurodevelopmental disorders,” Chamberlain says. “I think once we identify a specific therapy for an autism-like disorder such as Angelman syndrome, that’s a proof of principle that can be applied to other genetic forms of autism. I think then that raises the bar, that says we need to be able to look for the genetic causes of autism and be willing to look harder for genetic cause[s]. Because for each case where we can pinpoint a genetic cause, we can go back and [use] CRISPR technology, for instance, to design a specific therapy for those kids.”
Most versions of CRISPR-Cas9 technology can only work in one mode at a time, says Albert Cheng, an assistant professor at The Jackson Laboratory for Genomic Medicine in Farmington. Cheng has developed a modified version of CRISPR-Cas9 called Casilio that can operate in different modes on multiple genes at once.
“The original CRISPR-Cas is like the first cellphone: you can call but you can’t do other stuff,” he says. “[With] the new version of CRISPR-Cas9 we’re trying to make, you can now install apps, you can do different things on the genome at the same time, like a cutting-edge smartphone.”
Cheng is working on developing the Casilio platform so that it can allow for editing of dozens of genes at once. Ultimately he hopes “people can use it to edit, turn on, or turn off different genes at the same time. That’s very powerful because in a lot of biology and disease problems it’s not one gene that is affected but multiple, tens or hundreds of genes, and they are affected in different ways.”
Cheng is not working on a specific disease but rather on developing a platform that gives other researchers a more effective and accurate method of targeting specific genes. CRISPR-Cas9 is not yet perfect. Cheng likens existing CRISPR-Cas9 technology to the autocorrect on smartphones. “It sometimes gives you the wrong edit. There’s much work to be done to make it 100 percent accurate,” he says.
What other gene-editing technologies are being developed?
Glazer, from Yale, and W. Mark Saltzman, a Yale biomedical engineer, have designed nanoparticles that deliver synthetic DNA — the PNA molecules Ricciardi was preparing in the lab — and provide an alternative gene-editing platform to CRISPR-Cas9. This technology has shown promise in lab tests with mice and human cells in cystic fibrosis, sickle cell anemia and thalassemia, a genetic disorder that can cause anemia.
In mice with thalassemia, the PNA-carrying nanoparticles were able to repair about 5 percent of the target cells. CRISPR acts on 30 to 50 percent of target cells, but the Yale method has a major advantage because the off-target effects are 10,000- to 100,000-fold lower than CRISPR.
Using the nanoparticles on mice with cystic fibrosis, Glazer and his collaborators were able to elicit genetic changes, but not in enough cells to constitute a cure or treatment for the disease. “We didn’t really fix the disease, but we could detect gene editing, so it was sort of a first step,” he says.
He believes they are closer to developing a treatment for sickle cell anemia that could lead to human trials.
“We know from the work we did in thalassemia that we can get correction in the bone marrow in the range of 5 or 10 percent of the cells,” he says. “We’re not quite there with the sickle cell target, so we need to get up in that range or maybe in the 15 to 20 percent range, and I think once we do that it will be possible to start a clinical trial.”
How far away are gene-editing therapies in humans?
Chamberlain says, “From the research I’m aware of it seems like a long ways out, but I suspect that some companies are working on this in private. I think that field is so hot and moves so rapidly that I think the companies working on this are holding the cards close to their chest.”
Chamberlain has heard “rumors that a lab in Boston has miniaturized a CRISPR activator.” She adds, “The only reason to miniaturize it is to really make it so it’s therapeutically useful,” which means this lab likely has a proof-of-principle treatment in development.
Cheng says gene editing is now mostly used by scientists to model diseases and identify disease-causing genes, but that the most exciting aspect of the field is using it to “directly treat diseases.” He says there are many projects on the horizon. “There is a lot of work already done on animal studies and proof-of-principle studies to treat diseases. You can change one particular gene and make cells HIV resistant. … People have already used it to inactivate viral genes in the pig genome, so you can make organ transplants from pig to humans safer [and possible].”
Some of these future advancements may be thanks to work done in Connecticut.
For her Ph.D. thesis, Ricciardi is working with Glazer and Saltzman to perform in utero gene editing of developing fetuses.
“We’re doing this in mice now,” she says. “The idea is that you can prevent some of the disease manifestation or organ damage from occurring before the baby is born if you’re able to correct the genes early enough. That is a big step forward for gene editing because no one has corrected a gene in utero. People have worked on delivering components of genes but no one’s actually corrected a gene in utero — that’s what we’re excited about.”
She can’t disclose the specific conditions they’re working on, as the research has not yet been published, but she says the focus is on serious conditions that affect children. “These are diseases that make children sick. If a child had this disease they would be spending [a] significant amount of time in doctors’ offices receiving therapies that are treating the symptoms of their disease but not necessarily correcting the underlying genetic [cause] that’s causing the disease.”
Ricciardi says creating real-world treatments is what drew her to this area of research. “I wanted to work on a project that was translational, something that we were working on in the lab that applied to human disease.”