Magazine Article | May 4, 2015

Search For New Therapeutic Targets Turns To "Loss-Of- Function" DNA Variants

Source: Life Science Leader

By Cathy Yarbrough, Contributing Editor
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Fifteen years ago at a White House ceremony announcing the completion of the Human Genome Project’s draft of the human DNA sequence, President Bill Clinton told the audience of industry and academic leaders, government officials, and journalists that “today’s historic achievement is only a starting point. There is much hard work yet to be done.”

Some of that hard work was presented at the Future of Genomic Medicine (FoGM) VIII conference, March 5 and 6, in La Jolla, CA. More than 550 researchers and clinicians from 10 countries, including the U.S., attended the meeting, organized by the Scripps Translational Science Institute in La Jolla. The topics of the 25 conference presentations ranged from prenatal testing to President Barack Obama’s Precision Medicine Initiative.

Like other scientific meetings on genomics during the past decade, the FoGM conference demonstrated that, in retrospect, President Clinton’s comments were incredibly accurate. The socalled “promise” of the Human Genome Project — a bonanza of new and improved drugs for virtually every serious disease — has not yet been achieved because the human DNA sequence has proven to be stubbornly complex, much more indecipherable than researchers could have realized in 2000. There are obvious exceptions, of course, such as the FDA-approved drugs gefitinib and erlotinib that target genetic mutations common to many lung cancers. And, in diagnosis, “we are in the middle of a revolution in prenatal care,” said FoGM speaker Diana Bianchi, M.D., geneticist and neonatologist at Tufts University School of Medicine in Boston. Because of advances in genomics, prenatal genetic testing can now use blood samples obtained from pregnant patients rather than amniotic fluid, which is obtained by amniocentesis, a much more invasive procedure.

Since the launch of the Human Genome Project, scientists in both industry and academia have been searching for deleterious mutations, the DNA variants that significantly increase an individual’s risk for developing a chronic disease such as type 2 diabetes (T2D). A second category of human genetic mutations also provides potential targets for drug development. These are the naturally occurring loss-of-function (LOF) DNA variants that are protective against specific diseases without causing ill effects. “There is a lot we can learn from nature about disease prevention,” said Eric Topol, M.D., FoGM conference chairman and STSI director.

Although LOF gene variants were not a dominant theme of the FoGM meeting, their value as potential therapeutic targets stood out. Mark McCarthy, M.D., professor of diabetes at the University of Oxford, U.K., briefly spoke about the recent discovery of the LOF mutation in the gene SLC30A8 that protects individuals from T2D. Pfizer is investigating the LOF gene variant in T2D drug development. Scientists from Sangamo Biosciences and Regeneron Pharmaceuticals told how LOF gene variants have led to the design of two innovative therapeutics, one of which may be approved this summer by the FDA.

“You start with a naturally occurring variation, and then you aim to recapitulate it to create a disease-protective genotype and then a phenotype in a clinical setting,” said conference speaker and Sangamo team leader Fyodor Urnov, Ph.D. He and his team at Sangamo used a naturally occurring LOF variant in the CCR5 gene to design a very different therapeutic against the human immunodeficiency virus (HIV). Sangamo’s experimental therapeutic, SB-728-T, now in Phase 2 clinical trials, recapitulates the LOF protective genetic mutation that is estimated to occur in about 1 percent of the Caucasian population.

The CCR5 LOF mutation was detected several years ago when an HIV patient’s cancer was treated with a bone marrow transplant using donor cells. After the transplant, the patient’s viral load quickly dropped, and his T-cell count soared. The patient’s dramatic improvement was subsequently attributed to the LOF mutations in the CCR5 genes of the donor’s cells. The cells had two copies of the LOF mutation, one from each of the donor’s parents.

CCR5 genes code for the CCR5 protein receptors on the surface of T-cells. To invade T-cells, HIV first must lock onto these receptors. However, in individuals whose CCR5 genes have natural LOF mutations, the cell receptors are disabled. As a result, HIV cannot infect their T-cells, and they are naturally resistant to HIV infection. By using cutting-edge genome-editing laboratory technology, Sangamo scientists succeeded in inducing LOF mutations in the genomes of T-cells from HIV patients. Urnov explained that as a result of the editing, the CCR5 receptors on the T-cells were disabled, and the genome-edited T-cells did not become infected with HIV when experimentally exposed to the virus in laboratory cultures.

Thus far, more than 70 HIV patients have been experimentally treated with their own genome-edited T-cells. The edited cells are administered by infusion. “The treatment has been well tolerated,” Urnov said. Clinical trial results indicate that genome-edited T-cells can persist for as long as 250 days post-infusion. The T-cell counts remained high even in the subset of patients whose prescribed antiretroviral therapies were briefly discontinued for 12 weeks on the 28th day after infusion. Long-term viral control occurred in 24 patients, he added.

Sangamo’s HIV therapeutic is a proprietary technology that uses zinc finger nucleases (ZFNs) customized by the company’s researchers. In the clinical trials, thus far, ZFNs were used to edit the CCR5 genes of T-cells removed from the patients’ blood circulation. In an upcoming Phase 1 trial, Sangamo’s ZFNs will be employed to edit the genomes of stem cells harvested from HIV patients’ bone marrow. These stem cells are precursors of blood cells, T-cells, and other immune system cells. In addition to HIV, Sangamo has targeted sickle cell anemia, transfusion- dependent beta-thalassemia, and other hemoglobinopathies. The company’s scientists have tailored proprietary ZFNs for each of these diseases.

The clinical studies of Sangamo’s ZFNs for HIV are the first patient studies of a genome-editing therapeutic for any condition. Genome editing, which enables researchers to disable a targeted gene as well as precisely insert a DNA sequence into the genomes of human cells in laboratory cultures, is a “transformative, disrupting technology,” said conference speaker Keith Joung, M.D., Ph.D. With the technology, researchers also can create laboratory cell lines with the same characteristics of diseased patients’ cells. These cell lines are ideal for disease modeling and screening of experimental compounds, added Joung, associate professor of pathology, Harvard Medical School and cofounder of the new genome editing biotech company Editas Medicine. Joung also spoke about the epigenome-editing technologies that he and other researchers are employing to investigate the genomic factors that regulate human gene expression.

Another LOF genetic mutation that has led to a novel therapeutic is the variant of the PCSK9 gene, which normally encodes a protein whose actions help raise blood levels of low-density lipoprotein (LDL), the so-called “bad” cholesterol. Pfizer, Lilly, Amgen, Regeneron, and Sanofi are among the biopharmaceutical companies that have targeted the PCSK9 LDL as a therapeutic target. The closest to the finish line of achieving an FDA approval are Regeneron and Sanofi, which have been working together. Their PCSK9 inhibitor is a fully humanized mouse monoclonal antibody.

In January 2015, the two companies’ BLA (biologics license application) for their inhibitor, alirocumab, was designated for priority review by the FDA. The agency is expected to formally respond to the application in July 2015, said FoGM speaker George D. Yancopoulos, M.D., Ph.D., founding scientist and chief scientific officer of Regeneron. If approved by the FDA in July, alirocumab will be the first in the new class of PCSK9 inhibitors. The second-in-class may be Amgen’s PCSK9 inhibitor, evolocumab. The FDA is scheduled to respond to Amgen’s BLA in August 2015, said Yancopoulos. “Amgen has been on our heels the whole time,” he jokingly told the FoGM audience.

Several weeks after the FoGM meeting, researchers reported in the New England Journal of Medicine that alirocumab and evolocumab reduced by over 60 percent the LDL levels of heart disease patients in clinical trials. The results concurrently were presented at the American College of Cardiology’s annual meeting. PCSK9 inhibitors, administered by injection, are designed for patients with high blood levels of LDL cholesterol who cannot tolerate the cholesterol-lowering statin drugs or for whom the statins have been ineffective.

PCSK9 inhibitors originated with the 2003 discovery of a French geneticist who was studying the genetics of a family, many of whose members died from cardiovascular disease at an early age. The scientist linked the family’s high blood cholesterol levels to a gainof- function variant of the PCSK9 gene. Subsequently, U.S. researchers identified the LOF variant of the same gene in a subset of 300 African-Americans with very low levels of blood cholesterol, explained Yancopoulos.

"We think we’re getting pretty smart about understanding genomics and functional aspects of how the genome works, but we’re just scratching the surface."

NIH director


“Alirocumab is the first Regeneron drug that recapitulates an LOF genetic mutation, but hopefully not the last,” commented Yancopoulos. To identify other LOF drug targets, Regeneron in 2014 launched a first-of-its-kind collaboration with the Pennsylvania-based Geisinger Health System that eventually will include 100,000 patient volunteers. In addition to new LOF variants, the five-year program will search for the gain-of-function mutations that magnify an individual’s risk for developing disease.

The patient volunteers, whose identities are concealed from the company’s researchers, agree to allow their DNA to be sequenced and genotyped. Because Regeneron researchers have access to the volunteers’ electronic health records, they are able to search for patterns suggesting possible links between genetic factors, disease occurrence, and health outcomes. During the first year of the collaboration, almost 250 LOF genetic variants have been identified and are under study in Regeneron’s labs.

“The identification of new LOF genetic variants is one of many objectives of President Barack Obama’s $215 million Precision Medicine Initiative (PMI),” said NIH Director Francis Collins, M.D., Ph.D., who spoke at the FoGM conference. Government funding for the PMI, which was announced by President Obama in his State of the Union address in January 2015, will be included in the White House 2016 budget proposal to the U.S. Congress, said Collins, who added that the details of the initiative are “in the process of being defined.”

PMI’s centerpiece will be a study of 1 million Americans who will be asked to voluntarily provide blood samples for extensive genomic, metabolic, proteomic, and microbiomic testing. The test results, along with behavioral data gathered from smartphones, Fitbits, and other digital devices, will be linked to the patients’ electronic medical records. “This large-scale cohort will give us access to the deep information and power that we’ve not had before,” said Collins, who described the cohort as the “quantified self, multiplied by a million.” The “quantified self” refers to comprehensive self-monitoring with digital devices and other technologies.

Collins added that the longitudinal study will be a “phenomenal foundation platform for testing all manner of interventions,” including new drugs. Many of the volunteers will come from existing study cohorts such as the patient population of the Geisinger Health System.

President Obama’s new initiative could help unravel the complexities of the human genome. “Where we are right now, we think we’re getting pretty smart about understanding genomics and functional aspects of how the genome works,” said Collins. “But we’re just scratching the surface.” Referring to a future FoGM conference, Collins may have provided a clue about the many years of research that will be required to make genomic medicine a day-to-day reality. He said that if the speakers at the FoGM conference in 2027 review the presentations of the 2015 meeting, they would say, “We were really ignorant about so many things.”