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CRISPR/Cas9

Engineering a Better Way to Deliver Therapeutic Genes to Muscles

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Green adenovirus delivers therapeutic genes to muscles which glow green

Amid all the progress toward ending the COVID-19 pandemic, it’s worth remembering that researchers here and around the world continue to make important advances in tackling many other serious health conditions. As an inspiring NIH-supported example, I’d like to share an advance on the use of gene therapy for treating genetic diseases that progressively degenerate muscle, such as Duchenne muscular dystrophy (DMD).

As published recently in the journal Cell, researchers have developed a promising approach to deliver therapeutic genes and gene editing tools to muscle more efficiently, thus requiring lower doses [1]. In animal studies, the new approach has targeted muscle far more effectively than existing strategies. It offers an exciting way forward to reduce unwanted side effects from off-target delivery, which has hampered the development of gene therapy for many conditions.

In boys born with DMD (it’s an X-linked disease and therefore affects males), skeletal and heart muscles progressively weaken due to mutations in a gene encoding a critical muscle protein called dystrophin. By age 10, most boys require a wheelchair. Sadly, their life expectancy remains less than 30 years.

The hope is gene therapies will one day treat or even cure DMD and allow people with the disease to live longer, high-quality lives. Unfortunately, the benign adeno-associated viruses (AAVs) traditionally used to deliver the healthy intact dystrophin gene into cells mostly end up in the liver—not in muscles. It’s also the case for gene therapy of many other muscle-wasting genetic diseases.

The heavy dose of viral vector to the liver is not without concern. Recently and tragically, there have been deaths in a high-dose AAV gene therapy trial for X-linked myotubular myopathy (XLMTM), a different disorder of skeletal muscle in which there may already be underlying liver disease, potentially increasing susceptibility to toxicity.

To correct this concerning routing error, researchers led by Mohammadsharif Tabebordbar in the lab of Pardis Sabeti, Broad Institute of MIT and Harvard and Harvard University, Cambridge, MA, have now assembled an optimized collection of AAVs. They have been refined to be about 10 times better at reaching muscle fibers than those now used in laboratory studies and clinical trials. In fact, researchers call them myotube AAVs, or MyoAAVs.

MyoAAVs can deliver therapeutic genes to muscle at much lower doses—up to 250 times lower than what’s needed with traditional AAVs. While this approach hasn’t yet been tried in people, animal studies show that MyoAAVs also largely avoid the liver, raising the prospect for more effective gene therapies without the risk of liver damage and other serious side effects.

In the Cell paper, the researchers demonstrate how they generated MyoAAVs, starting out with the commonly used AAV9. Their goal was to modify the outer protein shell, or capsid, to create an AAV that would be much better at specifically targeting muscle. To do so, they turned to their capsid engineering platform known as, appropriately enough, DELIVER. It’s short for Directed Evolution of AAV capsids Leveraging In Vivo Expression of transgene RNA.

Here’s how DELIVER works. The researchers generate millions of different AAV capsids by adding random strings of amino acids to the portion of the AAV9 capsid that binds to cells. They inject those modified AAVs into mice and then sequence the RNA from cells in muscle tissue throughout the body. The researchers want to identify AAVs that not only enter muscle cells but that also successfully deliver therapeutic genes into the nucleus to compensate for the damaged version of the gene.

This search delivered not just one AAV—it produced several related ones, all bearing a unique surface structure that enabled them specifically to target muscle cells. Then, in collaboration with Amy Wagers, Harvard University, Cambridge, MA, the team tested their MyoAAV toolset in animal studies.

The first cargo, however, wasn’t a gene. It was the gene-editing system CRISPR-Cas9. The team found the MyoAAVs correctly delivered the gene-editing system to muscle cells and also repaired dysfunctional copies of the dystrophin gene better than the CRISPR cargo carried by conventional AAVs. Importantly, the muscles of MyoAAV-treated animals also showed greater strength and function.

Next, the researchers teamed up with Alan Beggs, Boston Children’s Hospital, and found that MyoAAV was effective in treating mouse models of XLMTM. This is the very condition mentioned above, in which very high dose gene therapy with a current AAV vector has led to tragic outcomes. XLMTM mice normally die in 10 weeks. But, after receiving MyoAAV carrying a corrective gene, all six mice had a normal lifespan. By comparison, mice treated in the same way with traditional AAV lived only up to 21 weeks of age. What’s more, the researchers used MyoAAV at a dose 100 times lower than that currently used in clinical trials.

While further study is needed before this approach can be tested in people, MyoAAV was also used to successfully introduce therapeutic genes into human cells in the lab. This suggests that the early success in animals might hold up in people. The approach also has promise for developing AAVs with potential for targeting other organs, thereby possibly providing treatment for a wide range of genetic conditions.

The new findings are the result of a decade of work from Tabebordbar, the study’s first author. His tireless work is also personal. His father has a rare genetic muscle disease that has put him in a wheelchair. With this latest advance, the hope is that the next generation of promising gene therapies might soon make its way to the clinic to help Tabebordbar’s father and so many other people.

Reference:

[1] Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Tabebordbar M, Lagerborg KA, Stanton A, King EM, Ye S, Tellez L, Krunnfusz A, Tavakoli S, Widrick JJ, Messemer KA, Troiano EC, Moghadaszadeh B, Peacker BL, Leacock KA, Horwitz N, Beggs AH, Wagers AJ, Sabeti PC. Cell. 2021 Sep 4:S0092-8674(21)01002-3.

Links:

Muscular Dystrophy Information Page (National Institute of Neurological Disorders and Stroke/NIH)

X-linked myotubular myopathy (Genetic and Rare Diseases Information Center/National Center for Advancing Translational Sciences/NIH)

Somatic Cell Genome Editing (Common Fund/NIH)

Mohammadsharif Tabebordbar (Broad Institute of MIT and Harvard and Harvard University, Cambridge, MA)

Sabeti Lab (Broad Institute of MIT and Harvard and Harvard University)

NIH Support: Eunice Kennedy Shriver National Institute of Child Health and Human Development; Common Fund


Could CRISPR Gene-Editing Technology Be an Answer to Chronic Pain?

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Active Neurons
Credit: iStock/Firstsignal

Gene editing has shown great promise as a non-heritable way to treat a wide range of conditions, including many genetic diseases and more recently, even COVID-19. But could a version of the CRISPR gene-editing tool also help deliver long-lasting pain relief without the risk of addiction associated with prescription opioid drugs?

In work recently published in the journal Science Translational Medicine, researchers demonstrated in mice that a modified version of the CRISPR system can be used to “turn off” a gene in critical neurons to block the transmission of pain signals [1]. While much more study is needed and the approach is still far from being tested in people, the findings suggest that this new CRISPR-based strategy could form the basis for a whole new way to manage chronic pain.

This novel approach to treating chronic pain occurred to Ana Moreno, the study’s first author, when she was a Ph.D. student in the NIH-supported lab of Prashant Mali, University of California, San Diego. Mali had been studying a wide range of novel gene- and cell-based therapeutics. While reading up on both, Moreno landed on a paper about a mutation in a gene that encodes a pain-enhancing protein in spinal neurons called NaV1.7.

Moreno read that kids born with a loss-of-function mutation in this gene have a rare condition known as congenital insensitivity to pain (CIP). They literally don’t sense and respond to pain. Although these children often fail to recognize serious injuries because of the absence of pain to alert them, they have no other noticeable physical effects of the condition.

For Moreno, something clicked. What if it were possible to engineer a new kind of treatment—one designed to turn this gene down or fully off and stop people from feeling chronic pain?

Moreno also had an idea about how to do it. She’d been working on repressing or “turning off” genes using a version of CRISPR known as “dead” Cas9 [2]. In CRISPR systems designed to edit DNA, the Cas9 enzyme is often likened to a pair of scissors. Its job is to cut DNA in just the right spot with the help of an RNA guide. However, CRISPR-dead Cas9 no longer has any ability to cut DNA. It simply sticks to its gene target and blocks its expression. Another advantage is that the system won’t lead to any permanent DNA changes, since any treatment based on CRISPR-dead Cas9 might be safely reversed.

After establishing that the technique worked in cells, Moreno and colleagues moved to studies of laboratory mice. They injected viral vectors carrying the CRISPR treatment into mice with different types of chronic pain, including inflammatory and chemotherapy-induced pain.

Moreno and colleagues determined that all the mice showed evidence of durable pain relief. Remarkably, the treatment also lasted for three months or more and, importantly, without any signs of side effects. The researchers are also exploring another approach to do the same thing using a different set of editing tools called zinc finger nucleases (ZFNs).

The researchers say that one of these approaches might one day work for people with a large number of chronic pain conditions that involve transmission of the pain signal through NaV1.7. That includes diabetic polyneuropathy, sciatica, and osteoarthritis. It also could provide relief for patients undergoing chemotherapy, along with those suffering from many other conditions. Moreno and Mali have co-founded the spinoff company Navega Therapeutics, San Diego, CA, to work on the preclinical steps necessary to help move their approach closer to the clinic.

Chronic pain is a devastating public health problem. While opioids are effective for acute pain, they can do more harm than good for many chronic pain conditions, and they are responsible for a nationwide crisis of addiction and drug overdose deaths [3]. We cannot solve any of these problems without finding new ways to treat chronic pain. As we look to the future, it’s hopeful that innovative new therapeutics such as this gene-editing system could one day help to bring much needed relief.

References:

[1] Long-lasting analgesia via targeted in situ repression of NaV1.7 in mice. Moreno AM, Alemán F, Catroli GF, Hunt M, Hu M, Dailamy A, Pla A, Woller SA, Palmer N, Parekh U, McDonald D, Roberts AJ, Goodwill V, Dryden I, Hevner RF, Delay L, Gonçalves Dos Santos G, Yaksh TL, Mali P. Sci Transl Med. 2021 Mar 10;13(584):eaay9056.

[2] Nuclease dead Cas9 is a programmable roadblock for DNA replication. Whinn KS, Kaur G, Lewis JS, Schauer GD, Mueller SH, Jergic S, Maynard H, Gan ZY, Naganbabu M, Bruchez MP, O’Donnell ME, Dixon NE, van Oijen AM, Ghodke H. Sci Rep. 2019 Sep 16;9(1):13292.

[3] Drug Overdose Deaths. Centers for Disease Control and Prevention.

Links:

Congenital insensitivity to pain (National Center for Advancing Translational Sciences/NIH)

Opioids (National Institute on Drug Abuse/NIH)

Mali Lab (University of California, San Diego)

Navega Therapeutics (San Diego, CA)

NIH Support: National Human Genome Research Institute; National Cancer Institute; National Institute of General Medical Sciences; National Institute of Neurological Disorders and Stroke


What A Year It Was for Science Advances!

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Science Breakthroughs of the Year 2020

At the close of every year, editors and writers at the journal Science review the progress that’s been made in all fields of science—from anthropology to zoology—to select the biggest advance of the past 12 months. In most cases, this Breakthrough of the Year is as tough to predict as the Oscar for Best Picture. Not in 2020. In a year filled with a multitude of challenges posed by the emergence of the deadly coronavirus disease 2019 (COVID-2019), the breakthrough was the development of the first vaccines to protect against this pandemic that’s already claimed the lives of more than 360,000 Americans.

In keeping with its annual tradition, Science also selected nine runner-up breakthroughs. This impressive list includes at least three areas that involved efforts supported by NIH: therapeutic applications of gene editing, basic research understanding HIV, and scientists speaking up for diversity. Here’s a quick rundown of all the pioneering advances in biomedical research, both NIH and non-NIH funded:

Shots of Hope. A lot of things happened in 2020 that were unprecedented. At the top of the list was the rapid development of COVID-19 vaccines. Public and private researchers accomplished in 10 months what normally takes about 8 years to produce two vaccines for public use, with more on the way in 2021. In my more than 25 years at NIH, I’ve never encountered such a willingness among researchers to set aside their other concerns and gather around the same table to get the job done fast, safely, and efficiently for the world.

It’s also pretty amazing that the first two conditionally approved vaccines from Pfizer and Moderna were found to be more than 90 percent effective at protecting people from infection with SARS-CoV-2, the coronavirus that causes COVID-19. Both are innovative messenger RNA (mRNA) vaccines, a new approach to vaccination.

For this type of vaccine, the centerpiece is a small, non-infectious snippet of mRNA that encodes the instructions to make the spike protein that crowns the outer surface of SARS-CoV-2. When the mRNA is injected into a shoulder muscle, cells there will follow the encoded instructions and temporarily make copies of this signature viral protein. As the immune system detects these copies, it spurs the production of antibodies and helps the body remember how to fend off SARS-CoV-2 should the real thing be encountered.

It also can’t be understated that both mRNA vaccines—one developed by Pfizer and the other by Moderna in conjunction with NIH’s National Institute of Allergy and Infectious Diseases—were rigorously evaluated in clinical trials. Detailed data were posted online and discussed in all-day meetings of an FDA Advisory Committee, open to the public. In fact, given the high stakes, the level of review probably was more scientifically rigorous than ever.

First CRISPR Cures: One of the most promising areas of research now underway involves gene editing. These tools, still relatively new, hold the potential to fix gene misspellings—and potentially cure—a wide range of genetic diseases that were once to be out of reach. Much of the research focus has centered on CRISPR/Cas9. This highly precise gene-editing system relies on guide RNA molecules to direct a scissor-like Cas9 enzyme to just the right spot in the genome to cut out or correct a disease-causing misspelling.

In late 2020, a team of researchers in the United States and Europe succeeded for the first time in using CRISPR to treat 10 people with sickle cell disease and transfusion-dependent beta thalassemia. As published in the New England Journal of Medicine, several months after this non-heritable treatment, all patients no longer needed frequent blood transfusions and are living pain free [1].

The researchers tested a one-time treatment in which they removed bone marrow from each patient, modified the blood-forming hematopoietic stem cells outside the body using CRISPR, and then reinfused them into the body. To prepare for receiving the corrected cells, patients were given toxic bone marrow ablation therapy, in order to make room for the corrected cells. The result: the modified stem cells were reprogrammed to switch back to making ample amounts of a healthy form of hemoglobin that their bodies produced in the womb. While the treatment is still risky, complex, and prohibitively expensive, this work is an impressive start for more breakthroughs to come using gene editing technologies. NIH, including its Somatic Cell Genome Editing program, continues to push the technology to accelerate progress and make gene editing cures for many disorders simpler and less toxic.

Scientists Speak Up for Diversity: The year 2020 will be remembered not only for COVID-19, but also for the very public and inescapable evidence of the persistence of racial discrimination in the United States. Triggered by the killing of George Floyd and other similar events, Americans were forced to come to grips with the fact that our society does not provide equal opportunity and justice for all. And that applies to the scientific community as well.

Science thrives in safe, diverse, and inclusive research environments. It suffers when racism and bigotry find a home to stifle diversity—and community for all—in the sciences. For the nation’s leading science institutions, there is a place and a calling to encourage diversity in the scientific workplace and provide the resources to let it flourish to everyone’s benefit.

For those of us at NIH, last year’s peaceful protests and hashtags were noticed and taken to heart. That’s one of the many reasons why we will continue to strengthen our commitment to building a culturally diverse, inclusive workplace. For example, we have established the NIH Equity Committee. It allows for the systematic tracking and evaluation of diversity and inclusion metrics for the intramural research program for each NIH institute and center. There is also the recently founded Distinguished Scholars Program, which aims to increase the diversity of tenure track investigators at NIH. Recently, NIH also announced that it will provide support to institutions to recruit diverse groups or “cohorts” of early-stage research faculty and prepare them to thrive as NIH-funded researchers.

AI Disentangles Protein Folding: Proteins, which are the workhorses of the cell, are made up of long, interconnected strings of amino acids that fold into a wide variety of 3D shapes. Understanding the precise shape of a protein facilitates efforts to figure out its function, its potential role in a disease, and even how to target it with therapies. To gain such understanding, researchers often try to predict a protein’s precise 3D chemical structure using basic principles of physics—including quantum mechanics. But while nature does this in real time zillions of times a day, computational approaches have not been able to do this—until now.

Of the roughly 170,000 proteins mapped so far, most have had their structures deciphered using powerful imaging techniques such as x-ray crystallography and cryo–electron microscopy (cryo-EM). But researchers estimate that there are at least 200 million proteins in nature, and, as amazing as these imaging techniques are, they are laborious, and it can take many months or years to solve 3D structure of a single protein. So, a breakthrough certainly was needed!

In 2020, researchers with the company Deep Mind, London, developed an artificial intelligence (AI) program that rapidly predicts most protein structures as accurately as x-ray crystallography and cryo-EM can map them [2]. The AI program, called AlphaFold, predicts a protein’s structure by computationally modeling the amino acid interactions that govern its 3D shape.

Getting there wasn’t easy. While a complete de novo calculation of protein structure still seemed out of reach, investigators reasoned that they could kick start the modeling if known structures were provided as a training set to the AI program. Utilizing a computer network built around 128 machine learning processors, the AlphaFold system was created by first focusing on the 170,000 proteins with known structures in a reiterative process called deep learning. The process, which is inspired by the way neural networks in the human brain process information, enables computers to look for patterns in large collections of data. In this case, AlphaFold learned to predict the underlying physical structure of a protein within a matter of days. This breakthrough has the potential to accelerate the fields of structural biology and protein research, fueling progress throughout the sciences.

How Elite Controllers Keep HIV at Bay: The term “elite controller” might make some people think of video game whizzes. But here, it refers to the less than 1 percent of people living with human immunodeficiency virus (HIV) who’ve somehow stayed healthy for years without taking antiretroviral drugs. In 2020, a team of NIH-supported researchers figured out why this is so.

In a study of 64 elite controllers, published in the journal Nature, the team discovered a link between their good health and where the virus has inserted itself in their genomes [3]. When a cell transcribes a gene where HIV has settled, this so-called “provirus,” can produce more virus to infect other cells. But if it settles in a part of a chromosome that rarely gets transcribed, sometimes called a gene desert, the provirus is stuck with no way to replicate. Although this discovery won’t cure HIV/AIDS, it points to a new direction for developing better treatment strategies.

In closing, 2020 presented more than its share of personal and social challenges. Among those challenges was a flood of misinformation about COVID-19 that confused and divided many communities and even families. That’s why the editors and writers at Science singled out “a second pandemic of misinformation” as its Breakdown of the Year. This divisiveness should concern all of us greatly, as COVID-19 cases continue to soar around the country and our healthcare gets stretched to the breaking point. I hope and pray that we will all find a way to come together, both in science and in society, as we move forward in 2021.

References:

[1] CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. Frangoul H et al. N Engl J Med. 2020 Dec 5.

[2] ‘The game has changed.’ AI triumphs at protein folding. Service RF. Science. 04 Dec 2020.

[3] Distinct viral reservoirs in individuals with spontaneous control of HIV-1. Jiang C et al. Nature. 2020 Sep;585(7824):261-267.

Links:

COVID-19 Research (NIH)

2020 Science Breakthrough of the Year (American Association for the Advancement of Science, Washington, D.C)


Discussing the Long Arc of Discovery with NIH’s Newest Nobelist

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Discussion with Dr. Harvey Alter

It’s been a tough year for our whole world because of everything that’s happening as a result of the coronavirus disease 2019 (COVID-19) pandemic. Yet there are bright spots that still shine through, and this week brought some fantastic news about NIH-supported researchers being named 2020 Nobel Prize Laureates for their pioneering work in two important fields: Chemistry and Physiology or Medicine.

In the wee hours of Wednesday morning, NIH grantee Jennifer A. Doudna, a biochemist at the University of California, Berkeley, got word that she and Emmanuelle Charpentier, a microbiologist at the Max Planck Institute for Infection Biology, Berlin, Germany, had won the 2020 Nobel Prize in Chemistry for developing the CRISPR/cas approach to genome editing. Doudna has received continuous NIH funding since 1997, mainly from the National Institute of General Medical Sciences and National Human Genome Research Institute.

The CRISPR/cas system, which consists of a short segment of RNA attached to the cas enzyme, provides the ability to make very precise changes in the sequence, or spelling, of the genetic instruction books of humans and other species. If used to make non-heritable edits in relevant tissues, such technology holds enormous potential to treat or even cure a wide range of devastating diseases, including thousands of genetic disorders where the DNA misspelling is precisely known.

Just two days before Doudna learned of her big award, a scientist who’s spent almost his entire career at the NIH campus in Bethesda, MD, received news that he too was getting a Nobel—the 2020 Nobel Prize in Physiology or Medicine. Harvey Alter, a senior scholar in the NIH Clinical Center’s Transfusion Medicine Department, was recognized for his contributions in identifying the potentially deadly hepatitis C virus. He shares this year’s prize with Michael Houghton, now with University of Alberta, Edmonton, and Charles M. Rice, The Rockefeller University, New York, who’s received continuous NIH funding since 1987, mainly from the National Institute of Allergy and Infectious Diseases.

In a long arc of discovery rooted in basic, translational, and clinical research that spanned several decades, Alter and his colleagues doggedly pursued biological clues that at first led to tests, then life-saving treatments, and, today, the very real hope of eradicating the global health threat posed by hepatitis C infections.

We at NIH are particularly proud of the fact that Alter is the sixth Nobel Prize winner—and the first in 26 years—to have done the entirety of his award-winning research in our Intramural Research Program. So, I jumped at the opportunity to talk with Harvey on NIH’s Facebook Live and Twitter chats just hours after he got the good news on Monday. Here’s a condensed version of our conversation, which took place on the NIH campus, but at a safe physical distance to minimize the risk of COVID-19 spread.

Collins: Harvey, let me start off by asking, how did you find out you’d won the Nobel Prize?

Alter: At 4:15 this morning. I was asleep and heard the telephone ringing. I ignored it. Five minutes later, I got another call. Now, I’m getting kind of perturbed. But I ignored it, thinking the call must be some kind of solicitation. Then, the phone rang a third time. I answered it, prepared to tell the person on the other end not to call me anymore. I heard a man’s voice say, “I’m the Secretary General of the Nobel Prize, calling you from Stockholm.” At that point, I just froze.

Collins: Did you think it might be a hoax?

Alter: No, I didn’t think it was a hoax. But I wasn’t expecting to win the prize. I knew about three years ago that I’d been on a Nobel list. But it didn’t happen, and I just forgot about it. Truthfully, I didn’t know that today was the day that the announcement was being made. The news came as a complete shock.

Collins: Please say a few words about viral hepatitis. What is it?

Alter: Sure. Viral hepatitis is an infection of the liver that causes inflammation and can lead to scarring, or cirrhosis. Early in my career, two viruses were known to cause the disease. One was the hepatitis A virus. You got it from consuming contaminated water or food. The second was the hepatitis B virus, which has a blood-borne transmission, typically from blood transfusions. In the 1970s, we realized that some other agent was causing most of the hepatitis from blood transfusions. Since it wasn’t A and it wasn’t B, we cleverly decided to call it: non-A, non-B. We did that because we hadn’t yet proven that the causative agent was a virus.

Collins: So, even though you screened donor units for the hepatitis B virus to eliminate tainted blood, people were still getting hepatitis from blood transfusions. How did you go about trying to solve this mystery?

Alter: The main thing was to follow patients prospectively, meaning forward in time. We drew a blood sample before they were transfused, and then serially afterwards. We saved those samples and also the donor samples to compare them. Using a liver function test, we found that 30 percent of patients who had open heart surgery at NIH prior to 1970 developed liver abnormalities indicative of hepatitis. That’s 1 in 3 people.

We then looked for the reasons. We found the main one was our source of blood. We were buying blood, which was then in short supply, from commercial laboratories. It turned out that their paid donors were engaging in high-risk behaviors [Note: like IV drug users sharing hypodermic needles]. We immediately stopped using these laboratories, and, through various other measures, we got the rate down to around 4 percent in 1987.

That’s when Michael Houghton, then at Chiron Corp. and a co-recipient of this year’s prize, cloned the virus. Think about it, he and his colleagues looked at 6 million clones and found just one that reacted with the convalescent serum of a patient with non-A, non-B. In other words, having contracted the virus, the patient already made antibodies against it that were present in the serum. If that one clone came from the virus, the antibodies in the serum would recognize it. They did, and Chiron then developed an assay to detect antibodies to the virus.

Collins: And that’s when they contacted you.

Alter: Yes, they wanted to use our panel of patient blood samples that had fooled a lot of people who claimed to have developed a non-A, non-B assay. Nobody else had “broken” this panel, but the Chiron Corp. did. We found that every case of non-A, non-B was really hepatitis C, the agent that they had cloned. Hepatitis C was the missing piece. As far as we could tell, there were no other agents beside hepatitis B and C that would result in transfusion transmission of the disease.

Collins: This story is clearly one of persistence. So, say something about persistence as an important characteristic of a scientist. You’re a great example of someone who was always looking out for opportunities that might not have seemed so promising at first.

Alter: I first learned persistence from Dr. Baruch Blumberg, my first NIH mentor who discovered the hepatitis B virus in 1967. [Note: Other NIH researchers identified the hepatitis A virus in 1977] The discovery started when we found this “Australian antigen,” a molecular structure that the immune system recognizes as foreign and attacks. It was a serendipitous finding that could have been easily just dropped. But he just kept at it, kept at it, kept at it. He had this famous wall where he diagrammed his hypotheses with all the contingencies if one worked or failed. Then, all of a sudden, the antigen was associated with hepatitis B. It became the basis of the hepatitis B vaccine, which is highly effective and used throughout the world. Dr. Blumberg won the Nobel Prize for his work on the hepatitis B virus in 1976.

Collins: Sometimes people look at NIH and ask why we don’t focus all of our efforts on curing a particular disease. I keep answering, ‘Wait a moment, we don’t know enough to know how to do that.’ What’s the balance that we ought to be seeking between basic research and clinical applications?

Alter: There is this tendency now to pursue highly directed research to solve a problem. That’s certainly how biopharma works. They want a payoff. The NIH is different. It’s a place where you can pursue your scientific interests, wherever they lead. The NIH leadership understands that the details of a problem often aren’t obvious at first. Researchers need to be allowed to observe things and then to pursue their leads as far as possible, with the understanding that not everything will work out. I think it’s very important to keep this basic research component in parallel with the more clinical applications. In the case of hepatitis C, it started as a clinical problem that led to a basic research investigation, which led back to a clinical problem. It was bedside-to-bench-to-bedside.

Collins: Are people still getting infected with hepatitis C?

Alter: Yes, hepatitis C remains a global problem. Seventy million people have contracted the virus, though the majority are generally asymptomatic, meaning they don’t get sick from it. Instead, they carry around the virus for decades without knowing it. That’s because the hepatitis C virus likes to persist, and our immune system doesn’t seem to be able to get rid of it easily.

However, some of those infected will have bad outcomes, such as cirrhosis or cancer of the liver. But there’s no way of knowing who will and who won’t get sick over time. The trick now is to identify people when they’re asymptomatic and without obvious disease.

That involves testing. We’re in a unique position with hepatitis C, where we have great tests that are highly sensitive and very specific to the virus. We also have great treatments. We can cure everybody who is tested and found to be positive.

Collins: People may be surprised to hear that. Here is a chronic viral illness, for which we actually have a cure. That’s come along fairly recently. Say a bit more about that—it’s such a great story of success.

Alter: For many years, the only treatment for hepatitis C was interferon, a very difficult treatment that initially had only about a 6 percent cure rate. With further progress, it got up to around 50 percent. But the big breakthrough came in the late 1990s when Gilead Corp., having the sequenced genome of the hepatitis C virus, deduced what it needs to replicate. If we know what it needs and we interfere with that, we can stop the replication. Gilead came out with a blockbuster drug that, now in combination with another drug, aims at two different sites on the virus and cures at least 98 percent of people. It’s an oral therapy taken for only 12 weeks, sometimes as little as 8 weeks, and with virtually no side-effects. It’s like a miracle drug.

Collins: What would you say to somebody who is thinking about becoming a scientist? How do you pick an area of research that will be right for you?

Alter: It’s a tough question. Medical research is very difficult, but there’s nothing more rewarding than doing something for patients and to see a good outcome like we had with hepatitis C.

The best path forward is to work for somebody who’s already an established investigator and a good teacher. Work in his or her lab for a few years and get involved in a project. I’ve learned not get into a lot of projects. Get into something where you can become the expert and pursue it.
The other thing is to collaborate. There’s no way that one person can do everything these days. You need too much technology and lots of different areas of expertise.

Collins: You took on a high-risk project in which you didn’t know that you’d find the answer. What’s the right balance between a project that you know will be productive, and something that might be risky, but, boy, if it works, could be transformative? How did you decide which of those paths to go?

Alter: I don’t think I decided. I just went! But there were interim rewards. Finding that the paid donors were bad was a reward and it had a big impact. And the different donor testing, decreasing the amount of blood [transfused], there were all kinds of steps along the way that gave you a reward. Now, did I think that there would be a treatment, an eradication of post-transfusion hepatitis at the end of my line? No, I didn’t.

And it wouldn’t have happened if it was only me. I just got the ball rolling. But it needed Houghton’s group. It needed the technology of Charlie Rice, a co-recipient of this year’s Nobel Prize. It needed joint company involvement. So, it required massive cooperation, and I have to say that here at NIH, Bob Purcell did most of the really basic work in his lab. Patrizia Farci, my closest collaborator, does things that I can’t do. You just need people who have a different expertise.

Collins: Harvey, it’s been maybe six hours since you found out that you won the Nobel Prize. How are you going to spend the rest of your day?

Alter: Well, I have to tell you a story that just happened. We had a press conference earlier today at NIH. Afterwards, I wanted to return to my NIH office and the easiest route was through the parking garage across the street from where we held the press conference. When I entered the garage, a security guard said, “You can’t come in, you haven’t been screened for COVID.” I assured him that I had been screened when I drove onto the NIH campus. He repeated that I had to go around to the front of the building to get screened.

Finally, I said to him, “Would it make any difference if I told you that I won the Nobel Prize today?” He replied, ‘That’s nice, but you must go around to the front of the building.’” So, winning the Nobel doesn’t give you immediate rewards!

Collins: Let me find that security guard and give him a bonus for doing a good job. Well, Harvey, will there be that trip to Stockholm coming up in December?

Alter: Not this year. I’ve heard that they will invite us to Stockholm next year to receive the award. But there’s going to be something in the US. I don’t know what it will be. I’ll invite you.

Collins: I will be glad to take part in the celebration. Well, Harvey, I really want to thank you for taking some time on this special day to reflect on your career and how the Nobel Committee came calling at 4:30 this morning. We’re really proud of you!

Alter: Thank you.

Links:

Hepatitis C (National Institute of Diabetes and Digestive and Kidney Diseases/NIH)

The Nobel Assembly at Karolinska Institutet has today decided to award the 2020 Nobel Prize in Physiology or Medicine jointly to Harvey J. Alter, Michael Houghton and Charles M. Rice for the discovery of Hepatitis C virus,” Nobel Prize announcement, October 5,2020.

Harvey Alter (Clinical Center/NIH)

The Road Not Taken, or How I Learned to Love the Liver: A Personal Perspective on Hepatitis History” Alter HJ, Hepatology. 2014 Jan;59(1):4-12.

Reflections on the History of HCV: A Posthumous Examination.” Alter HJ, Farci P, Bukh J, Purcell RH. Clinical Liver Disease, 15:1, Feb 2020.

Is Elimination of Hepatitis B and C a Pipe Dream or Reality?” Alter HJ, Chisari FV. Gastroenterology. 2019 Jan;156(2):294-296.

Michael Houghton (University of Alberta, Edmonton)

Charles Rice (The Rockefeller University, New York)

What is genome editing? (National Human Genome Research Institute/NIH)

Jennifer Doudna (University of California, Berkeley)

Emmanuelle Charpentier (Max Planck Institute for Infection Biology, Berlin, Germany)


Gene-Editing Advance Puts More Gene-Based Cures Within Reach

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Prime Editing
Caption: The prime editing system (left) contains three parts: two enzymes, Cas9 and reverse transcriptase, and an engineered guide RNA, pegRNA. Unlike regular CRISPR gene editing, prime editing nicks just one strand of the DNA molecule (right) and then uses RNA and reverse transcriptase to direct highly targeted changes to a cell’s DNA. Credit: Broad Institute of MIT and Harvard, Cambridge, MA.

There’s been tremendous excitement recently about the potential of CRISPR and related gene-editing technologies for treating or even curing sickle cell disease (SCD), muscular dystrophy, HIV, and a wide range of other devastating conditions. Now comes word of another remarkable advance—called “prime editing”—that may bring us even closer to reaching that goal.

As groundbreaking as CRISPR/Cas9 has been for editing specific genes, the system has its limitations. The initial version is best suited for making a double-stranded break in DNA, followed by error-prone repair. The outcome is generally to knock out the target. That’s great if eliminating the target is the desired goal. But what if the goal is to fix a mutation by editing it back to the normal sequence?

The new prime editing system, which was described recently by NIH-funded researchers in the journal Nature, is revolutionary because it offers much greater control for making a wide range of precisely targeted edits to the DNA code, which consists of the four “letters” (actually chemical bases) A, C, G, and T [1].

Already, in tests involving human cells grown in the lab, the researchers have used prime editing to correct genetic mutations that cause two inherited diseases: SCD, a painful, life-threatening blood disorder, and Tay-Sachs disease, a fatal neurological disorder. What’s more, they say the versatility of their new gene-editing system means it can, in principle, correct about 89 percent of the more than 75,000 known genetic variants associated with human diseases.

In standard CRISPR, a scissor-like enzyme called Cas9 is used to cut all the way through both strands of the DNA molecule’s double helix. That usually results in the cell’s DNA repair apparatus inserting or deleting DNA letters at the site. As a result, CRISPR is extremely useful for disrupting genes and inserting or removing large DNA segments. However, it is difficult to use this system to make more subtle corrections to DNA, such as swapping a letter T for an A.

To expand the gene-editing toolbox, a research team led by David R. Liu, Broad Institute of MIT and Harvard, Cambridge, MA, previously developed a class of editing agents called base editors [2,3]. Instead of cutting DNA, base editors directly convert one DNA letter to another. However, base editing has limitations, too. It works well for correcting four of the most common single letter mutations in DNA. But at least so far, base editors haven’t been able to make eight other single letter changes, or fix extra or missing DNA letters.

In contrast, the new prime editing system can precisely and efficiently swap any single letter of DNA for any other, and can make both deletions and insertions, at least up to a certain size. The system consists of a modified version of the Cas9 enzyme fused with another enzyme, called reverse transcriptase, and a specially engineered guide RNA, called pegRNA. The latter contains the desired gene edit and steers the needed editing apparatus to a specific site in a cell’s DNA.

Once at the site, the Cas9 nicks one strand of the double helix. Then, reverse transcriptase uses one DNA strand to “prime,” or initiate, the letter-by-letter transfer of new genetic information encoded in the pegRNA into the nicked spot, much like the search-and-replace function of word processing software. The process is then wrapped up when the prime editing system prompts the cell to remake the other DNA strand to match the new genetic information.

So far, in tests involving human cells grown in a lab dish, Liu and his colleagues have used prime editing to correct the most common mutation that causes SCD, converting a T to an A. They were also able to remove four DNA letters to correct the most common mutation underlying Tay-Sachs disease, a devastating condition that typically produces symptoms in children within the first year and leads to death by age four. The researchers also used their new system to insert new DNA segments up to 44 letters long and to remove segments at least 80 letters long.

Prime editing does have certain limitations. For example, 11 percent of known disease-causing variants result from changes in the number of gene copies, and it’s unclear if prime editing can insert or remove DNA that’s the size of full-length genes—which may contain up to 2.4 million letters.

It’s also worth noting that now-standard CRISPR editing and base editors have been tested far more thoroughly than prime editing in many different kinds of cells and animal models. These earlier editing technologies also may be more efficient for some purposes, so they will likely continue to play unique and useful roles in biomedicine.

As for prime editing, additional research is needed before we can consider launching human clinical trials. Among the areas that must be explored are this technology’s safety and efficacy in a wide range of cell types, and its potential for precisely and safely editing genes in targeted tissues within living animals and people.

Meanwhile, building on all these bold advances, efforts are already underway to accelerate the development of affordable, accessible gene-based cures for SCD and HIV on a global scale. Just last month, NIH and the Bill & Melinda Gates Foundation announced a collaboration that will invest at least $200 million over the next four years toward this goal. Last week, I had the chance to present this plan and discuss it with global health experts at the Grand Challenges meeting Addis Ababa, Ethiopia. The project is an unprecedented partnership designed to meet an unprecedented opportunity to address health conditions that once seemed out of reach but—as this new work helps to show—may now be within our grasp.

References:

[1] Search-and-replace genome editing without double-strand breaks or donor DNA. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR. Nature. Online 2019 October 21. [Epub ahead of print]

[2] Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Nature. 2016 May 19;533(7603):420-424.

[3] Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. Nature. 2017 Nov 23;551(7681):464-471.

Links:

Tay-Sachs Disease (Genetics Home Reference/National Library of Medicine/NIH)

Sickle Cell Disease (National Heart, Lung, and Blood Institute/NIH)

Cure Sickle Cell Initiative (NHLBI)

What are Genome Editing and CRISPR-Cas9? (National Library of Medicine/NIH)

Somatic Cell Genome Editing Program (Common Fund/NIH)

David R. Liu (Harvard, Cambridge, MA)

NIH Support: National Institute of Allergy and Infectious Diseases; National Human Genome Research Institute; National Institute for General Medical Sciences; National Institute of Biomedical Imaging and Bioengineering; National Center for Advancing Translational Sciences


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