Dark Genome

Harnessing the Dark Genome: New Approach Greatly Improves Cancer T-Cell Therapy

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An artistic rendering of CRISPR-enhanced T-cells attacking a tumor. Research has shown that the gene-editing system CRISPR can modulate T-cell behavior to make them better cancer killers without actually editing any of their genes. Credit: Ella Maru Studio

A CRISPR-based platform has discovered numerous genes with the potential to enhance T-cell therapies for cancer treatment.

Researchers at Duke University have advanced CRISPR technologies for large-scale analysis of gene functions in human immune cells. They found that a single key regulator in the genome can reprogram a vast network of genes in T cells, significantly boosting their ability to kill cancer cells.

The master regulator is called BATF3 and is one of several genes that the researchers identified and tested for improving T-cell therapies. These targets, and the methods developed to identify, test, and manipulate them, could make any of the T-cell cancer therapies currently in use and under development more potent. Combined with other advances, the platform could also enable generalized, off-the-shelf versions of the therapy and expansion into other disease areas such as autoimmune disorders.

The results were recently published in the journal Nature Genetics.

Challenges in T-Cell Therapy and New Methodologies

T-cell therapy is a decade-old approach to treating cancer. More recent versions involve reprogramming the immune system’s primary soldiers to seek and destroy cancerous cells that they might otherwise overlook. Many companies are working to enhance the technology, mostly through the use of genetic engineering techniques that instruct the T cells on how to identify cancerous cells and make them more effective at destroying them.

There are currently six FDA-approved T-cell therapies for specific leukemias, lymphomas, and multiple myeloma. Their approaches, however, do not currently fare well when applied to solid tumors, although there are hints of success in certain studies. Solid tumors often present large physical barriers for the T cells to overcome, and the sheer number and density of cancer cells presenting targets can lead to “T-cell exhaustion,” wearing the attackers out to the point that they are not able to mount an antitumor response.

“In some cases, T-cell therapy works like a miracle drug, but in most others, it hardly works at all,” said Charles Gersbach, the John W. Strohbehn Distinguished Professor of Biomedical Engineering at Duke. “We are looking for generic solutions that can make these cells better across the board by reprogramming their gene regulation software, rather than rewriting or damaging their genetic hardware. This demonstration is a crucial step toward overcoming a major hurdle to getting T-cell therapy to work in more patients across a greater range of cancer types.”

Gersbach and his laboratory have spent the past several years developing a method that uses a version of the gene-editing technology CRISPR-Cas9 to explore and modulate genes without cutting them. Instead, it makes changes to the structures that package and store the DNA, affecting the activity level of the accompanying genes.

BATF3’s Role in Enhancing T-Cell Function

Sean McCutcheon, a Ph.D. candidate working in Gersbach’s lab and lead author of the study, focused on regions of this ‘dark genome’ that change as T cells transition between states, such as functional versus exhausted. He identified 120 genes that encode “master regulators,” which are responsible for the activity levels of many other genes. Using the CRISPR platform, he dialed the activity levels of these targets both up and down to see how they affected other known markers of T cell function.

While several promising candidates emerged, one of the most promising was a gene called BATF3. When McCutcheon subsequently delivered BATF3 directly to the T cells, there were thousands of tweaks to the packaging structure of the T cells’ DNA, and this correlated with increased potency and resistance to exhaustion.

“A known barrier to using T cells to fight cancer is that they tend to get ‘tired’ over time and lose their ability to kill cancer cells,” McCutcheon said. “We’re identifying manipulations that make T cells stronger and more resilient by mimicking naturally occurring cell states that work well in clinical products.”

The researchers put BATF3 through a battery of tests. The most interesting results came when they overexpressed BATF3 in T cells programmed to attack human breast cancer tumors in a mouse model. While the standard-of-care T-cell therapy struggled to slow tumor growth, the exact same dose of T-cells engineered with BATF3 completely eradicated the tumors.

Broad Implications and Future Research

While the results with BATF3 are exciting to Gersbach, McCutcheon, and the rest of the group, they are even more enthusiastic about the general success of the methodology to identify and modulate master regulators to improve therapeutic performance, which they have been developing for the better part of a decade. They can now readily profile master regulators of T cell fitness using any T cell source or cancer model and under various experimental conditions that mimic the clinical setting.

For example, in the last part of this study, McCutcheon screened T cells, with or without BATF3, while using CRISPR to remove every other master regulator of gene expression — more than 1,600 regulators in total. This led to the discovery of a whole new set of factors that could be targeted alone or in combination with BATF3 to increase the potency of T-cell therapy.

“This study focused in-depth on one particular target identified by these CRISPR screens, but now that Sean and the team have the whole discovery engine up and running, we can do this over and over again for different models and tumor types,” Gersbach said. “This study suggests many strategies for applying this approach to enhance T-cell therapy, from using a patient’s own T cells to having a bank of generalized T cells for a wide variety of cancers. We hope that these technologies can be generally applicable across all strategies.”

The Progress and Promise of Gene Editing

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Earlier this year, a report prepared for the National Academies urged caution in developing the gene-editing technology known as CRISPR-Cas9, but stopped short of calling for an outright ban. Click here to read MedPage Today’s original report on the Academies’ position. In this follow-up, we review further developments with CRISPR and its regulation.

New technologies such as the CRISPR-Cas9 offer the possibility of altering an individual’s genome, or even a generation’s genome.

Jennifer Doudna, PhD, a geneticist and professor at the University of California Berkeley and the Howard Hughes Medical Institute, created CRISPR in collaboration with Emmanuelle Charpentier, PhD, of Umea University in Sweden, in 2012.

Out of fear the technology could be misused, Doudna advocated a worldwide moratorium on gene editing that involved heritable changes.

Thus far, no researchers have publicly stated that they have made germline alterations in a human embryo with the intent of nurturing it to birth. But over the past year they have inched closer.

In August, researchers at Oregon Health & Science University in Portland, Oregon, led by biologist Shoukhrat Mitalipov, PhD, for the first time in the U.S. demonstrated the potential to edit human embryo DNA to prevent a congenital heart condition known as hypertrophic cardiomyopathy, which may cause heart failure or sudden death.

Then in October, the New Scientist reported that the CRISPR method was showing promise across a range of diseases in animal studies, including in muscular dystrophy and liver disease.

 Most of the research involved ex vivoexperiments — removing cells, editing them in a lab and then replacing them.

While this process is “relatively easy” for immune cells or blood stem cells, “this isn’t possible with most bodily tissues,” noted the New Scientist’s Michael LePage.

Matthew Porteus, MD, PhD, associate professor of pediatrics at Stanford University and an NAM committee member, told a Senate, Health Education, Labor and Pensions Committee in November that the best approach for other conditions such as congenital blindness and muscular dystrophy likely involves in vivo gene editing.

Regarding other conditions studied through ex vivo experiments, Porteus said his lab developed a method for correcting mutations of sickle cells in patients’ stem cells. If a cure is found, it might take only a few “tweaks” to then find a cure for other illnesses, such as severe combined immunodeficiency, he noted.

He anticipates seeing multiple CRISPR-Cas9 clinical trials in the U.S. or Europe in the next 12-18 months, Porteus added.

What’s Next for Gene Editing?

Asked about the most notable breakthroughs in the field right now, R. Alta Charo, JD, co-chair of the Committee on Human Gene Editing and a professor at the University of Wisconsin in Madison, spoke of “the developing capacity to do epigenetic editing,” speaking on her own behalf, in an email to MedPage Today.

“[I]t offers the prospect of making beneficial changes that, because they are reversible, in many cases will pose fewer risks,” she said.

As another benefit, this form of gene editing could be used to respond to conditions that stem from a “constellation of genetic factors” rather than a single mutation, reported Wired.

Researchers have already begun testing epigenetic editing in mice for diseases such as diabetes, acute kidney disease, and muscular dystrophy, Wired noted.

“Successful somatic gene therapy” and the OHSU study “pending confirmation by the scientific community” are the most notable breakthroughs of the year, said Jeffrey Kahn, PhD, MPH, director of the Johns Hopkins Berman Institute of Bioethics in Baltimore, and a member of the NAM committee, in an email to MedPage Today.

However, he noted that pre-implantation genetic diagnosis could have replaced gene editing in the OHSU study. In other words, the researchers ignored one of the NAM committee’s key criteria for heritable gene editing: lack of a reasonable alternative.

Others disagreed.

Because the study did not involve a pregnancy or birth “it constitutes purely laboratory research” and would be “permissible” under committee guidelines, said Charo.

“An emerging area in gene editing is harnessing these new precision engineering tools to edit regions of the genome outside of genes,” said Neville Sanjana, PhD, a core faculty member at the New York Genome Center and a professor at New York University, in an email, responding to the same question.

The ‘Dark Genome’ Emerges as Target

Gene editing tools can help to translate these regulatory and noncoding variants, those outside of the genes — less than 2% are actually in the genes themselves.

This area is sometimes referred to as the “dark genome.”

“Most of our genome is actually this ‘noncoding’ DNA and not in genes (less than 2% is in genes). We understand very little about how this noncoding DNA works and how changes in the sequence (primary sequence — not epigenome) results in changes in gene expression and disease,” said Sanjana.

Sanjana also highlighted the approval of “a multitude” of new gene therapies by the FDA for conditions such as congenital blindness, spinal muscular atrophy and different hemophilias, which he said has also generated a lot of excitement.

Although some gene therapies have been around since the 1990s, not all involve gene editing. However, the approvals represent progress, he noted.

“It is clear that this new modality of therapy — adding back a missing or damaged gene — will open new avenues of medicine,” said Sanjana.

“[I]t is a matter of time before gene editing tools are also part of the gene therapy arsenal to aid in curing disease for which we currently have no therapies,” he added.

As always, oversight will remain important to this process.

“The challenge is to find the ‘just right’ regulatory approach for what are new, emerging, and controversial biotechnologies such as gene editing tools. That often requires some tweaking to get right, and I hope that there is willingness to engage in the discussion necessary to find the appropriate balance of control with a path for innovation,” wrote Kahn.