For years, scientists have been manipulating genes in animal and human cells to gain a better understanding of how genomes work, with an eye toward developing improved medical treatments. Gene editing, in particular, involves using nucleases – enzymes that essentially act as “molecular scissors” – to cut DNA strands and insert, delete, or replace a specific piece or sequence of DNA.
Until recently, the complexity of gene-editing techniques has limited scientists’ ability to fully leverage this tool in basic and clinical research. Now, evolving and easier-to-use genome-editing platforms are accelerating the pace of scientific research and may help generate promising new therapies for a range of diseases, including genetic disorders, cancer, and blood disorders such as hemophilia, sickle cell anemia, and immunodeficiencies.
Modifying DNA within hematopoietic stems cells (HSCs) has the potential to alter blood cells or the immune system. What’s more, it is conceivable that improved outcomes in patients with certain genetic disorders could be achieved through a single, or shortened, course of treatment.
Despite the progress that is being made and the enthusiasm about potential new therapies, gene editing is not always efficient or reproducible on a large scale, and can cause unintended genetic changes, or “off-target effects.” This problem raises important questions about the long-term safety of gene editing, an issue that needs to be resolved before the approach is routinely used to treat patients.
Recognizing the potential of genome editing to correct genetic flaws that cause many inherited blood disorders and other diseases, the American Society for Hematology (ASH) is helping to advance this emerging technology. On July 14 and 15, 2016, ASH hosted a Workshop on Genome Editing in Washington, DC, that brought together more than 150 leading clinical and laboratory-based scientists, funders, and regulators to discuss this cutting-edge technology. (Notably, this workshop focused on somatic cell editing, not germline editing.)
ASH Clinical News spoke with program co-chairs Mitchell Weiss, MD, PhD, the Arthur Nienhuis Endowed Chair in Hematology at St. Jude Children’s Research Hospital, in Memphis, Tennessee, J. Keith Joung, MD, PhD, professor of pathology at Harvard Medical School and pathologist at Massachusetts General Hospital in Boston, Massachusetts, and keynote speaker Stuart Orkin, MD, the David G. Nathan Professor of Pediatrics at Harvard Medical School, to learn more about the state of genome editing and highlights from the meeting.
Gene editing has become a powerful laboratory research tool for modeling diseases. By manipulating the genome of various organisms, scientists are now able to develop new cell lines and animal models, which will greatly enhance the quality and pace of basic and translational research.
According to Dr. Weiss, “the major discoveries in this area came from basic research studies of protein structure, plants, and bacteria. Without basic research, there would be no translational research and, for this reason, we must pursue, support, and fund basic research with the same vigor and enthusiasm [or more] as translational research.”
Technological Advances Driving Rapid Progress
A recurring theme at the meeting was the rapid pace with which these technologies are being developed, refined, and put into practice. “Our ability to manipulate the genome precisely and easily is accelerating at a remarkable pace,” said Dr. Weiss.
Dr. Joung agreed, commenting “It is very exciting that there is such rapid progress and the number of diseases being studied.”
Several gene-editing platforms are being used in basic and clinical research, many of which were extensively reviewed at the meeting, including:
- zinc finger nuclease (ZFNs)
- transcription activator-like effector nucleases (TALENs)
- clustered regularly interspaced short palindromic repeats (CRISPR-Cas9)
ZFNs, TALENs, and mega-TALs are artificial proteins designed to bind and cleave specific DNA sequences in the genome. These complex proteins are difficult to engineer in most laboratory settings, which has limited their use in academic research. However, biopharmaceutical companies are using ZFNs in pre-clinical trials for beta-thalassemia and sickle cell anemia, and early clinical trials are underway in hemophilia and HIV. TALENs are used to modify allogeneic donor T cells to treat different types of cancers.
CRISPR-Cas9 is a relatively newer gene-editing platform that received widespread attention and enthusiasm, partly because the technology is relatively easy to implement and more accessible to academic researchers. CRISPR-Cas9 has two components: a strand of guide RNA (gRNA) that locates a specific string of letters in a DNA sequence, and the Cas9 nuclease, which acts as the DNA cutting tool.
“It is undetermined which technology is going to work the best,” said Dr. Weiss. “Most likely different ones are going to work best for different applications.”
The Unknown Consequences of Off-Target Effects
Of course, gene editing can also result in unintended modifications at other points along the genome outside of the targeted DNA sequence, a phenomenon called “off-target effects.” This is a leading concern of researchers, clinicians, and drug regulators involved with gene-modulation therapies.
“We can identify off-target mutations that occur at a high frequency, but people are worried about rare ones that escape detection and may give cells growth or survival advantages that promote cancer,” said Dr. Weiss. “We don’t know quite how to deal with this yet.”
Researchers are developing methods that allow them to define where and how frequently off-target mutations are occurring.
“We need to be able to quantify the risks of off-target effects – when they happen, where they happen, and how often they happen,” said Dr. Joung, adding that his lab has been able to create more sensitive methods for off-target detection and make improvements on the Cas9 enzyme (known as “high-fidelity” variants) that can reduce off-target mutations to undetectable levels.
Dr. Joung cautions that no treatment is completely free of side effects and, at the end of the day, although off-target effects should be assessed and mitigated as best as possible, a risk-benefit analysis will need to be undertaken for each therapy.
A Novel Way of Changing DNA
Some researchers are even thinking outside the “nuclease” box. David R. Liu, PhD, professor of chemistry and chemical biology at Harvard University, presented a new approach for genome editing called “targeted base editing” that can change a single letter or base in the DNA.
Unlike other platforms that use nucleases to break DNA, Dr. Liu’s technique enzymatically converts one base into another (e.g., cytosine to uracil). In particular, this approach may have potential benefits for genetic disorders that arise from single point mutations, such as sickle cell disease.
“People are excited about the possibilities afforded by not having to break the DNA,” explained Dr. Joung.
“This is another unique strategy for editing genes,” added Dr. Orkin.
Intersection of Targeted Immunotherapy and Genome Editing
According to Dr. Weiss, one area for which gene editing may have an immediate and practical impact is facilitating immunotherapy for cancer. “T cells are easier to edit than HSCs, so it’s going to be more feasible initially,” explained Dr. Weiss.
While current immunotherapies (such as chimeric antigen receptor T-cell or CAR T-cell therapy) typically involve genetically modifying a patient’s own immune cells to recognize and destroy cancer cells, the use of gene editing may make it possible to produce allogeneic T cells from donors that can be used “off the shelf” in cancer patients. A clinical trial using the TALENs platform to enhance CAR T cells to treat acute myeloid leukemia is underway, Dr. Orkin noted.
“Editing allows one to have much more flexibility in what one can do to modify those cells and increase their potency,” he said. “The worlds of gene editing and cellular therapy were on separate tracks, but now they have converged.”
Despite the tremendous advances that have been achieved, there are a number of barriers to realizing the value of gene editing in research and clinical medicine.
Optimizing the modification of HSCs remains a major challenge. While it may be possible to achieve a high rate of modification in a small sample of cells, often this rate is difficult to replicate in large cell samples in animal models – and may be even more challenging to reproduce in the clinical setting.
“The trick is engineering the system to guide your nuclease where you want it to go,” Dr. Weiss explained. “Then you have to get it into the cells efficiently and make sure your results are well controlled.”
In addition, it is unknown what levels of HSC correction and bone marrow reconstitution are clinically meaningful for treating blood disorders. Dr. Joung said gaining a better understanding of these parameters needs to be a major focus of ongoing research for each disease.
Putting it All Together
Looking to the future, these experts believe that gene editing holds much promise, but they don’t expect conventional hematologic therapies to become obsolete.
“In some form, genome editing will be an aspect of standard care for some disorders in the future, but not ‘the’ standard of care,” Dr. Weiss said. “It’s a high-tech form of medicine that is very expensive and has potential dangers; some disorders have more simple fixes. It’s not going to replace useful drugs in the near future and it won’t be available in underdeveloped countries for a long time.”
Nevertheless, researchers and clinicians are enthusiastic about the possibilities for improved gene-editing techniques to provide a better understanding of the genetics of hemoglobinopathies and other diseases and lay the groundwork for developing better treatments.
“My hope is that we’ll see a number of these efforts leading to therapeutics that will have benefits for patients,” said Dr. Joung. “The scale and speed at which this is happening is very exciting because it makes you think that there is a strong possibility that many of these approaches will actually make a difference for patients sometime in the near future.”
Dr. Orkin shared this optimism. “It takes time to translate this type of science,” he said. “Looking ahead the technology is only going to get better. There is no question that over the next three to five years there will be clinical applications that are likely to be successful.”