Gene Editing May Offer Clues, Future Treatments for Kidney Disease

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Using an array of cutting edge tools and techniques, researchers around the country have achieved an incredible feat. They have learned to grow living 3D, kidney-like structures called kidney organoids in the laboratory using human cells.

“What we are trying to do with the kidney organoids is not only grow new kidney tissue, but learn fundamental things about how kidneys work,” explained Benjamin Freedman, PhD, an assistant professor at the University of Washington in Seattle.

These kidney organoids are grown from human stem cells, which can be coaxed into recreating some of the structures found in human kidneys. A powerful gene editing tool called CRISPR is often being used to insert kidney-disease–linked mutations into organoids allowing scientists to study how mutations contribute to disease.

Together these technologies promise to drive major advances in the understanding of normal kidney function, what goes wrong in kidney disease, and how it might be remedied. With further advances, the technology might one day allow scientists to grow transplantable kidney tissue in the laboratory, alleviating the shortage of kidneys for transplantation. But these potential advances are not without controversy. The CRISPR gene editing technology and its potential to modify human genes has sparked an ethical debate and led for some calls for limits on its use.

A powerful tool

Prior to the development of kidney organoids, scientists struggled to find a way to study living kidney tissue in the laboratory. For example, kidney tissue collected from the body quickly loses its structure under laboratory conditions and such tissue can’t be used to recreate disease progression, Freedman said.

“The models we’ve had haven’t been able to recreate the complexity of the kidney,” explained Freedman. But kidney organoids, while still much more primitive than a real kidney, are complex enough to recreate some of the kidney’s key features. The CRISPR technique makes these organoids even more valuable by allowing scientists to customize their genetics.

Scientists have been cutting genes out of DNA or inserting genes into DNA using various techniques for 2 decades, said Benjamin Humphreys, MD, PhD, chief of the division of nephrology at Washington University School of Medicine. “But that technology was cumbersome and required a high level of expertise,” Humphreys explained. It also was expensive. A single experiment using older gene editing techniques might cost $500 to $5000 dollars, while CRISPR costs just $30 (Miyagi A, et al. J Am Soc Nephrol 2016; 27:2940–2947).

The CRISPR technique commandeers a defense mechanism bacteria use to ward off viruses. In bacteria, CRISPR are repetitive sections of DNA that are used to store genes from viruses that have previously infected the bacteria. These viral DNA sequences allow the bacteria to seek out and destroy those sequences in future viral attackers. Scientists have learned to insert genes they want to target into these repeated sections of bacterial DNA to use this defense mechanism to find a gene in human DNA. When the CRISPR finds the targeted gene it uses an enzyme called Cas9 to cut the DNA and remove the target gene. It can also be used to replace the targeted gene with another one.

“The power of CRISPR is that it is a highly robust, precise, and cheap way to change genome sequences,” Humphreys said.

It is so easy to use that any laboratory can use it, and one of its fastest growing applications in kidney disease research is in developing customized organoids, Humphreys said. For example, scientists have used CRISPR to insert the mutated gene that causes polycystic kidney disease into the stem cells used to grow kidney organoids and these organoids grow cysts (Freedman BS, et al. Nat Comm 2015; 6:8715). The process can be carefully studied in the laboratory to better understand how the mutated gene causes this to happen and to test drugs that might stop or reverse the process, Freedman explained.

Hundreds of miniature organoids can also be produced and used to test the effects of numerous drugs simultaneously.

“You can do it on a scale you couldn’t do in animal models,” Freedman said.

Mice can also be genetically engineered to have mutations linked to kidney disease, but there are drawbacks to trying to study human diseases in mice. Mice are different from humans genetically and physiologically so they may not respond the same way humans will.

“Many findings in mice don’t carry over in humans,” explained Joseph Bonventre, MD, PhD, chief of the renal unit and director of the bioengineering division at Brigham and Women’s hospital in Boston. “They are good models, but there are situations where mouse models are not ideal.”

Organoids also open the door to personalized studies or drug testing. A kidney organoid could be grown with a genetic mutation thought to be causing a particular patient’s kidney disease. That would allow scientists to verify whether the suspect mutation, in fact, causes the disease and, if so, what might be done to modify its effects, according to Freedman.

“All of us have many mutations that could look like they could be causing disease,” explained Freedman. But sometimes the wrong culprit has been identified. Additionally, organoids would allow scientists to systematically test what various genes do in the kidney to find previously unsuspected genes that might contribute to kidney disease.

“We are at the dawn of the genetics era for kidney disease,” Freedman said. “Not only can we discover the genes—we will be able to fix those genes. It will take us into the next era of being able to understand kidney disease and how to treat it.”

Drawing the line

The ease of using CRISPR and its power to alter human genetics has, however, raised some ethical concerns about the use of the technology. For example, could the technology be used to create “designer babies” or lead to unexpected harms?

To address these emerging concerns, the National Academies of Sciences in 2015 hosted a summit and convened a committee of international research, regulatory, and ethics experts to address the need for oversight of such research. The committee released its report in mid-February 2017. The report clearly draws a line arguing that use of CRISPR in research should be limited to studies that aim to develop interventions that treat or prevent disease or disability, and should not be used to enhance humans.

“That has long been a concern [with gene editing],” said Jeffrey Kahn, PhD, MPH, director of the Johns Hopkins Berman Institute of Bioethics in Baltimore and member of the committee. “The tools are now better such that potential enhancement uses are closer in terms of the ability to do it. It’s more pressing.”

Drawing that line won’t always be easy, Kahn noted. Some applications of the technology will clearly fall into the treatment and prevention realm, for example, using CRISPR to edit the mutated genes that cause muscular dystrophy to boost muscle in affected individuals. But he noted that if the US Food and Drug Administration (FDA) approved such treatments it might be hard to prevent them from being used to boost muscle in individuals without the disease.

“It’s hard for the FDA to do that,” Kahn said.

For research using gene editing tools on most cell types, the committee found that existing safeguards and oversight of human research are sufficient. But the committee argued more oversight and discussion is necessary before gene editing of reproductive cells, which could result in genetic alterations in subsequent generations. Additionally, the committee argued that gene editing that could be inherited should only be done under a limited set of circumstances, for example, only when no other option for having a healthy genetic child is possible. It’s a more permissive stance than the call for a complete moratorium on inheritable gene editing that emerged from the 2015 meeting.

“What the report did is open up the door a crack to the possibility that under strict restrictions scientists could alter human embryos in select circumstances,” Humphreys said. “They made it very clear that a tremendous amount of research and discussion needs to take place.”

Although it is possible such technology could be applied to human embryos with mutations that cause kidney disease, Freedman thought it was unlikely to be used for this purpose.

“I don’t see [CRISPR] germ line editing being of major utility in kidney disease,” Freedman said.

He explained that already parents affected by polycystic kidney disease could use in vitro fertilization and have their embryos screened for disease-causing mutations and choose not to have affected embryos implanted. But he said it is important to discuss these potential applications.

“It is good to have the conversations and to keep them in perspective with the general moral and ethical challenges that face society,” Freedman said.

Kahn and his colleagues hope their report provides guidance for governments around the world grappling with how to regulate these emerging tools. But he noted it is just the start of the process. Additional international meetings to discuss these issues are scheduled to take place in Bejing and the United Kingdom.

“It’s the beginning of that conversation,” Kahn said. “It’s a global discussion that’s ongoing.”

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Potential treatments

Most ongoing work using CRISPR is still in the preclinical phase, but many in the field expect CRISPR-driven treatments for kidney disease may be on the horizon.

“We are in a time of very rapidly advancing knowledge concerning ways to manipulate and potentially correct genetic disease,” Humphreys said.

Currently, clinical trials are underway using CRISPR technology to edit mutations that cause blood diseases. Some genetic kidney diseases might also be targeted for treatment with CRISPR, noted Humphreys. Mutations that cause polycystic kidney disease might be one potential target for such gene therapy. Another potential target might be mutations in the APOL1 gene that contribute to kidney failure in African Americans. But first some major technical challenges must be overcome.

There is currently no efficient way to deliver CRISPR to the billions of cells in the kidney, explained Freedman. There are also still safety concerns that need to be fixed; for example, occasionally CRISPRs can miss their target and disrupt the wrong gene.

“We know gene editing technologies are getting better, but they do have off-target effects,” Freedman said. We don’t want to inadvertently cause disease when trying to cure disease.”

Another potential use of CRISPR is to enable scientists to grow transplantable kidneys in animals. For example, at a very early stage in development CRISPR could be used to turn off the genes that help grow a pig’s kidney, then human cells could be transplanted and coaxed into growing a human kidney.

“It’ll be interesting to see how close you can get to growing a human organ in an animal,” said Freedman. “It could be a powerful source of organs.”

But doing that won’t be easy. The animal’s immune system is likely to attack the growing organ and a human’s immune system will likely reject an organ with traces of animal cells, Freedman said.

There are also concerns that transplanting organs grown in pigs into humans could transmit retroviruses embedded in the pig’s DNA to humans, noted Humphreys. But one laboratory has shown that CRISPR can be used to inactivate these viruses (Yang L, et al. Science 2015; 350:1101–1104). The CRISPR technology might also be used to edit immune genes in the pig or humans to prevent immune reactions, he said.

Organoids themselves might one day offer a source of kidney tissue for transplant. The vision would be to harvest cells from a patient with kidney disease, use CRISPR to correct any disease-causing mutation, then grow healthy transplantable kidney tissue in the laboratory. This could mitigate the need for powerful immunosuppressive drugs since the cells would be the patient’s own and less likely to trigger rejection.

“In the long term, we would like to grow functional tissues and implant them back into the patient from which the organoid cells were derived,” Freedman said.

But Bonventre noted much work is still needed to develop organoids that integrate vasculature, nervous system cells, and immune cells.

“We need to have additional breakthroughs,” he said.

In the shorter term, Bonventre suggested that hybrid devices combining laboratory-grown tissue with mechanical systems might offer a rudimentary replacement kidney that is better than dialysis, even if it doesn’t completely replace all of the kidney’s abilities. Medications could be used to control potassium or pH, for example.

“A combination of engineering and cellular systems will get us there in a way that requires little medication support,” Bonventre said. He said with enough resources he predicts there could be significant progress toward hybrid kidneys over the next 5 to 10 years.

Alhough much work remains before gene editing or transplantation of laboratory-grown tissue can be used to treat patients, scientists working in the field are optimistic that research using these technologies will lead to new therapies.

“In the long run, both organoids and editing will lead to new medications that are more effective and safer than transplants or dialysis,” Freedman said.

May 2017 (Vol. 9, Number 5)