Posted on by Dr. Francis Collins
Despite continued progress in treatment and prevention, lung cancer remains our nation’s leading cause of cancer death. In fact, more Americans die of lung cancer each year than of breast, colon, and prostate cancers combined [1,2]. While cigarette smoking is a major cause, lung cancer also occurs in non-smokers. I’m pleased to report discovery of what we hope will be a much-needed drug target for a highly aggressive, difficult-to-treat form of the disease, called small cell lung cancer (SCLC).
Using gene-editing technology to conduct a systematic, large-scale search for druggable vulnerabilities in certain types of cancer cells grown in lab dishes, NIH-funded researchers recently identified a metabolic pathway that appears to play a key role in SCLC. What makes this news even more encouraging is drugs that block this pathway already exist. That includes one in clinical testing for other types of cancer, and another that’s FDA-approved and has been safely used for more than 20 years to treat people with rheumatoid arthritis.
The new work comes from the lab of Tyler Jacks, Massachusetts Institute of Technology (MIT), Cambridge. The Jacks lab, which is dedicated to understanding the genetic events that lead to cancer, develops mouse models engineered to carry the same genetic mutations that turn up in human cancers.
In work described in Science Translational Medicine, the team, co-led by Leanne Li and Sheng Rong Ng, applied CRISPR gene-editing tools to cells grown from some of their mouse models. Aiming high in terms of scale, researchers used CRISPR to knock out systematically, one by one, each of about 5,000 genes in cells from the SCLC mouse model, as well in cells from mouse models of other types of lung and pancreatic cancers. They looked to see what gene knockouts would slow down or kill the cancer cells, because that would be a good indication that the protein products of these genes, or the pathways they mediated, would be potential drug targets.
Out of those thousands of genes, one rose to the top of the list. It encodes an enzyme called DHODH (dihydroorotate dehydrogenase). This enzyme plays an important role in synthesizing pyrimidine, which is a major building block in DNA and RNA. Cytosine and thymine, the C and T in the four-letter DNA code, are pyrimidines; so is uracil, the U in RNA that takes the place of T in DNA. Because cancer cells are constantly dividing, there is a continual need to synthesize new DNA and RNA molecules to support the production of new daughter cells. And that means, unlike healthy cells, cancer cells require a steady supply of pyrimidine.
It turns out that the SCLC cells have an unexpected weakness relative to other cancer cells: they don’t produce as much pyrimidine. As a result, the researchers found blocking DHODH left the cells short on pyrimidine, leading to reduced growth and survival of the cancer.
This was especially good news because DHODH-blocking drugs, including one called brequinar, have already been tested in clinical trials for other cancers. In fact, brequinar is now being explored as a potential treatment for acute myeloid leukemia.
Might brequinar also hold promise for treating SCLC? To explore further, the researchers looked again to their genetic mouse model of SCLC. Their studies showed that mice treated with brequinar lived about 40 days longer than control animals. That’s a significant survival benefit in this system.
Brequinar treatment appeared to work even better when combined with other approved cancer drugs in mice that had SCLC cells transplanted into them. Further study in mice carrying SCLC tumors derived from four human patients added to this evidence. Two of the four human tumors shrunk in mice treated with brequinar.
Of course, mice are not people. But the findings suggest that brequinar or another DHODH blocker might hold promise as a new way to treat SCLC. While more study is needed to understand even better how brequinar works and explore potentially promising drug combinations, the fact that this drug is already in human testing for another indication suggests that a clinical trial to explore its use for SCLC might happen more quickly.
More broadly, the new findings show the promise of gene-editing technology as a research tool for uncovering elusive cancer targets. Such hard-fought discoveries will help to advance precise approaches to the treatment of even the most aggressive cancer types. And that should come as encouraging news to all those who are hoping to find new answers for hard-to-treat cancers.
 Cancer Stat Facts: Lung and Bronchus Cancer (National Cancer Institute/NIH)
 Key Statistics for Lung Cancer (American Cancer Society)
 Identification of DHODH as a therapeutic target in small cell lung cancer. Li L, Ng SR, Colón CI, Drapkin BJ, Hsu PP, Li Z, Nabel CS, Lewis CA, Romero R, Mercer KL, Bhutkar A, Phat S, Myers DT, Muzumdar MD, Westcott PMK, Beytagh MC, Farago AF, Vander Heiden MG, Dyson NJ, Jacks T. Sci Transl Med. 2019 Nov 6;11(517).
Small Cell Lung Cancer Treatment (NCI/NIH)
Tyler Jacks (Massachusetts Institute of Technology, Cambridge)
NIH Support: National Cancer Institute
Posted on by Dr. Francis Collins
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 .
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.
 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]
 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.
 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.
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
Posted on by Dr. Francis Collins
If used to make non-heritable genetic changes, CRISPR gene-editing technology holds tremendous promise for treating or curing a wide range of devastating disorders, including sickle cell disease, vision loss, and muscular dystrophy. Early efforts to deliver CRISPR-based therapies to affected tissues in a patient’s body typically have involved packing the gene-editing tools into viral vectors, which may cause unwanted immune reactions and other adverse effects.
Now, NIH-supported researchers have developed an alternative CRISPR delivery system: nanocapsules. Not only do these tiny, synthetic capsules appear to pose a lower risk of side effects, they can be precisely customized to deliver their gene-editing payloads to many different types of cells or tissues in the body, which can be extremely tough to do with a virus. Another advantage of these gene-editing nanocapsules is that they can be freeze-dried into a powder that’s easier than viral systems to transport, store, and administer at different doses.
In findings published in Nature Nanotechnology , researchers, led by Shaoqin Gong and Krishanu Saha, University of Wisconsin-Madison, developed the nanocapsules with specific design criteria in mind. They would need to be extremely small, about the size of a small virus, for easy entry into cells. Their surface would need to be adaptable for targeting different cell types. They also had to be highly stable in the bloodstream and yet easily degraded to release their contents once inside a cell.
After much hard work in the lab, they created their prototype. It features a thin polymer shell that’s easily decorated with peptides or other ingredients to target the nanocapsule to a predetermined cell type.
At just 25 nanometers in diameter, each nanocapsule still has room to carry cargo. That cargo includes a single CRISPR/Cas9 scissor-like enzyme for snipping DNA and a guide RNA that directs it to the right spot in the genome for editing.
In the bloodstream, the nanocapsules remain fully intact. But, once inside a cell, their polymer shells quickly disintegrate and release the gene-editing payload. How is this possible? The crosslinking molecules that hold the polymer together immediately degrade in the presence of another molecule, called glutathione, which is found at high levels inside cells.
The studies showed that human cells grown in the lab readily engulf and take the gene-editing nanocapsules into bubble-like endosomes. Their gene-editing contents are then released into the cytoplasm where they can begin making their way to a cell’s nucleus within a few hours.
Further study in lab dishes showed that nanocapsule delivery of CRISPR led to precise gene editing of up to about 80 percent of human cells with little sign of toxicity. The gene-editing nanocapsules also retained their potency even after they were freeze-dried and reconstituted.
But would the nanocapsules work in a living system? To find out, the researchers turned to mice, targeting their nanocapsules to skeletal muscle and tissue in the retina at the back of eye. Their studies showed that nanocapsules injected into muscle or the tight subretinal space led to efficient gene editing. In the eye, the nanocapsules worked especially well in editing retinal cells when they were decorated with a chemical ingredient known to bind an important retinal protein.
Based on their initial results, the researchers anticipate that their delivery system could reach most cells and tissues for virtually any gene-editing application. In fact, they are now exploring the potential of their nanocapsules for editing genes within brain tissue.
I’m also pleased to note that Gong and Saha’s team is part of a nationwide consortium on genome editing supported by NIH’s recently launched Somatic Cell Genome Editing program. This program is dedicated to translating breakthroughs in gene editing into treatments for as many genetic diseases as possible. So, we can all look forward to many more advances like this one.
 A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing. Chen G, Abdeen AA, Wang Y, Shahi PK, Robertson S, Xie R, Suzuki M, Pattnaik BR, Saha K, Gong S. Nat Nanotechnol. 2019 Sep 9.
Saha Lab (University of Wisconsin-Madison)
Shaoqin (Sarah) Gong (University of Wisconsin-Madison)
NIH Support: National Eye Institute; National Institute of General Medical Sciences; National Institute of Neurological Disorders and Stroke; National Heart, Lung, and Blood Institute; Common Fund
Posted on by Dr. Francis Collins
Nanoparticles hold great promise for delivering next-generation therapeutics, including those based on CRISPR gene editing tools. The challenge is how to guide these tiny particles through the bloodstream and into the right target tissues. Now, scientists are enlisting some surprising partners in this quest: magnetic bacteria!
First a bit of background. Discovered in the 1960s during studies of bog sediments, “magnetotactic” bacteria contain magnetic, iron-rich particles that enable them to orient themselves to the Earth’s magnetic fields. To explore the potential of these microbes for targeted delivery of nanoparticles, the NIH-funded researchers devised the ingenious system you see in this fluorescence microscopy video. This system features a model blood vessel filled with a liquid that contains both fluorescently-tagged nanoparticles (red) and large swarms of a type of magnetic bacteria called Magnetospirillum magneticum (not visible).
At the touch of a button that rotates external magnetic fields, researchers can wirelessly control the direction in which the bacteria move through the liquid—up, down, left, right, and even “freestyle.” And—get this—the flow created by the synchronized swimming of all these bacteria pushes along any nearby nanoparticles in the same direction, even without any physical contact between the two. In fact, the researchers have found that this bacteria-guided system delivers nanoparticles into target model tissues three times faster than a similar system lacking such bacteria.
How did anyone ever dream this up? Most previous attempts to get nanoparticle-based therapies into diseased tissues have relied on simple diffusion or molecular targeting methods. Because those approaches are not always ideal, NIH-funded researchers Sangeeta Bhatia, Massachusetts Institute of Technology, Cambridge, MA, and Simone Schürle, formerly of MIT and now ETH Zurich, asked themselves: Could magnetic forces be used to propel nanoparticles through the bloodstream?
As a graduate student at ETH Zurich, Schürle had worked to develop and study tiny magnetic robots, each about the size of a cell. Those microbots, called artificial bacterial flagella (ABF), were designed to replicate the movements of bacteria, relying on miniature flagellum-like propellers to move them along in corkscrew-like fashion.
In a study published recently in Science Advances, the researchers found that the miniature robots worked as hoped in tests within a model blood vessel . Using magnets to propel a single microbot, the researchers found that 200-nanometer-sized polystyrene balls penetrated twice as far into a model tissue as they did without the aid of the magnet-driven forces.
At the same time, others in the Bhatia lab were developing bacteria that could be used to deliver cancer-fighting drugs. Schürle and Bhatia wished they could direct those microbial swarms using magnets as they could with the microbots. That’s when they learned about the potential of M. magneticum and developed the experimental system demonstrated in the video above.
The researchers’ next step will be to test their magnetic approach to drug delivery in a mouse model. Ultimately, they think their innovative strategy holds promise for delivering nanoparticles carrying a wide range of therapeutic payloads right to a tumor, infection, or other diseased tissue. It’s yet another example of how basic research combined with outside-the-box thinking can lead to surprisingly creative solutions with real potential to improve human health.
 Synthetic and living micropropellers for convection-enhanced nanoparticle transport. Schürle S, Soleimany AP, Yeh T, Anand GM, Häberli M, Fleming HE, Mirkhani N, Qiu F, Hauert S, Wang X, Nelson BJ, Bhatia SN. Sci Adv. 2019 Apr 26;5(4):eaav4803.
What are genome editing and CRISPR-Cas9? (National Library of Medicine/NIH)
Sangeeta Bhatia (Massachusetts Institute of Technology, Cambridge, MA)
Simone Schürle-Finke (ETH Zurich, Switzerland)
NIH Support: National Cancer Institute; National Institute of General Medical Sciences
Posted on by Dr. Francis Collins
When I volunteered to serve as a physician at a hospital in rural Nigeria more than 25 years ago, I expected to treat a lot of folks with infectious diseases, such as malaria and tuberculosis. And that certainly happened. What I didn’t expect was how many people needed care for type 2 diabetes (T2D) and the health problems it causes. Surprisingly, these individuals were generally not overweight, and the course of their illness seemed different than in the West.
The experience inspired me to join with other colleagues at Howard University, Washington, DC, to help found the Africa America Diabetes Mellitus (AADM) study. It aims to uncover genomic risk factors for T2D in Africa and, using that information, improve understanding of the condition around the world.
So, I’m pleased to report that, using genomic data from more than 5,000 volunteers, our AADM team recently discovered a new gene, called ZRANB3, that harbors a variant associated with T2D in sub-Saharan Africa . Using sophisticated laboratory models, the team showed that a malfunctioning ZRANB3 gene impairs insulin production to control glucose levels in the bloodstream.
Since my first trip to Nigeria, the number of people with T2D has continued to rise. It’s now estimated that about 8 to 10 percent of Nigerians have some form of diabetes . In Africa, diabetes affects more than 7 percent of the population, more than twice the incidence in 1980 .
The causes of T2D involve a complex interplay of genetic, environmental, and lifestyle factors. I was particularly interested in finding out whether the genetic factors for T2D might be different in sub-Saharan Africa than in the West. But at the time, there was a dearth of genomic information about T2D in Africa, the cradle of humanity. To understand complex diseases like T2D fully, we need all peoples and continents represented in the research.
To begin to fill this research gap, the AADM team got underway and hasn’t looked back. In the latest study, led by Charles Rotimi at NIH’s National Human Genome Research Institute, in partnership with multiple African diabetes experts, the AADM team enlisted 5,231 volunteers from Nigeria, Ghana, and Kenya. About half of the study’s participants had T2D and half did not.
As reported in Nature Communications, their genome-wide search for T2D gene variants turned up three interesting finds. Two were in genes previously linked to T2D risk in other human populations. The third involved a gene that codes for ZRANB3, an enzyme associated with DNA replication and repair that had never been reported in association with T2D.
To understand how ZRANB3 might influence a person’s risk for developing T2D, the researchers turned to zebrafish (Danio rerio), an excellent vertebrate model for its rapid development. The researchers found that the ZRANB3 gene is active in insulin-producing beta cells of the pancreas. That was important to know because people with T2D frequently have reduced numbers of beta cells, which compromises their ability to produce enough insulin.
The team next used CRISPR/Cas9 gene-editing tools either to “knock out” or reduce the expression of ZRANB3 in young zebrafish. In both cases, it led to increased loss of beta cells.
Additional study in the beta cells of mice provided more details. While normal beta cells released insulin in response to high levels of glucose, those with suppressed ZRANB3 activity couldn’t. Together, the findings show that ZRANB3 is important for beta cells to survive and function normally. It stands to reason, then, that people with a lower functioning variant of ZRANB3 would be more susceptible to T2D.
In many cases, T2D can be managed with some combination of diet, exercise, and oral medications. But some people require insulin to manage the disease. The new findings suggest, particularly for people of African ancestry, that the variant of the ZRANB3 gene that one inherits might help to explain those differences. People carrying particular variants of this gene also may benefit from beginning insulin treatment earlier, before their beta cells have been depleted.
So why wasn’t ZRANB3 discovered in the many studies on T2D carried out in the United States, Europe, and Asia? It turns out that the variant that predisposes Africans to this disease is extremely rare in these other populations. Only by studying Africans could this insight be uncovered.
More than 20 years ago, I helped to start the AADM project to learn more about the genetic factors driving T2D in sub-Saharan Africa. Other dedicated AADM leaders have continued to build the research project, taking advantage of new technologies as they came along. It’s profoundly gratifying that this project has uncovered such an impressive new lead, revealing important aspects of human biology that otherwise would have been missed. The AADM team continues to enroll volunteers, and the coming years should bring even more discoveries about the genetic factors that contribute to T2D.
 ZRANB3 is an African-specific type 2 diabetes locus associated with beta-cell mass and insulin response. Adeyemo AA, Zaghloul NA, Chen G, Doumatey AP, Leitch CC, Hostelley TL, Nesmith JE, Zhou J, Bentley AR, Shriner D, Fasanmade O, Okafor G, Eghan B Jr, Agyenim-Boateng K, Chandrasekharappa S, Adeleye J, Balogun W, Owusu S, Amoah A, Acheampong J, Johnson T, Oli J, Adebamowo C; South Africa Zulu Type 2 Diabetes Case-Control Study, Collins F, Dunston G, Rotimi CN. Nat Commun. 2019 Jul 19;10(1):3195.
 Diabetes mellitus in Nigeria: The past, present and future. Ogbera AO, Ekpebegh C. World J Diabetes. 2014 Dec 15;5(6):905-911.
 Global report on diabetes. Geneva: World Health Organization, 2016. World Health Organization.
Diabetes (National Institute of Diabetes ad Digestive and Kidney Diseases/NIH)
Diabetes and African Americans (Department of Health and Human Services)
Why Use Zebrafish to Study Human Diseases (Intramural Research Program/NIH)
Charles Rotimi (National Human Genome Research Institute/NIH)
NIH Support: National Human Genome Research Institute; National Institute of Diabetes and Digestive and Kidney Diseases; National Institute on Minority Health and Health Disparities