Abstract
“Ideally, we want to be able to turn on and off the transferred genes at a specific time,” DKFZ virologist Dirk Nettelbeck, Ph.D., said in a statement.
Dr. Nettelbeck and colleagues pinched their idea from nature, which has already evolved riboswitches that in bacteria, for example, regulate gene expression in response to the binding of metabolites, second messengers, or toxic agents. Their artificial construct comprised a synthetic ligand-dependent self-cleaving ribozyme, inserted into the 5′ and/or 3′ untranslated region (UTR) of the transcription unit. Its design effectively keeps expression of the transgene off in the presence of the exogenous trigger, in this case theophylline, but allows its expression in the absence of theophylline.
The team successfully tested their aptazyme switches in adenoviral vectors, adeno-associated viruses, and oncolytic adenoviruses, and demonstrated that the switch allowed up to a 10-fold increase in transgene expression.
“This was the first proof that RNA switches work in viruses at all,” Dr. Nettelbeck said. “The construction of RNA switches is extremely variable. Once the technology is fully developed, we will be able to better equip and regulate viruses for many therapeutic applications.”
The researchers detailed their work in the journal Nucleic Acids Research, in a paper titled “Synthetic riboswitches for external regulation of genes transferred by replication-deficient and oncolytic adenoviruses (Ketzer et al., 2012).”
Writing for the court in a two-to-one decision, Circuit Judge Alan Lourie on August 16 upheld a lower court's 2010 denial of patent eligibility for Myriad's method claims directed to comparing or analyzing DNA sequences using the genes: “Such claims include no transformative steps and cover only patent-ineligible abstract, mental steps,” the judges wrote in their decision. (Read the decision at
That denial sided with part of the 2010 decision by the U.S. District Court for the Southern District of New York. But two other parts of the lower court's decision were overturned by the appellate majority. It found that the BRCA1 and BRCA2 genes represented “a nonnaturally occurring composition of matter,” and thus were patentable under § 101 of Title 35, U.S. Code.
Using that reasoning, the appellate majority also upheld Myriad's method claim to screening potential cancer therapeutics via changes in cell growth rates of transformed cells, rejecting the district court's finding that it was directed to a patent-ineligible scientific principle.
The appellate majority's most recent decision echoed a 2011 ruling by the same court. Circuit Judge Kimberly A. Moore concurred in part with Lourie, while Circuit Judge William Bryson dissented from the part of the decision holding BRCA1 and BRCA2 as patent-eligible: “The court's decision, if sustained, will likely have broad consequences, such as preempting methods for whole-genome sequencing.”
Bryson sided with plaintiffs in the original case Association for Molecular Pathology v. U.S. Patent and Trademark Office. The American Civil Liberties Union is counsel-of-record to AMP and coplaintiffs, which advocate a ban on gene patenting. They argue that gene patenting stifles basic scientific inquiry, and is therefore a violation of the spirit and letter of the intellectual property clause of Article I, § 8, of the U.S. Constitution.
Myriad rejects that argument and contends that patenting is vital to recouping development costs. The company has said it spent $500 million over 17 years on its BRACAnalysis test before breaking even.
Penn agreed to grant Novartis an exclusive worldwide license to technologies used in an ongoing trial of patients with chronic lymphocytic leukemia (CLL), as well as future CAR-based therapies developed. Novartis agreed to pay Penn royalties and additional payments tied to undisclosed milestones.
Announced on August 6, a year to the month when after a Penn research team published promising results in the New England Journal of Medicine (Porter et al., 2011) and Science Translational Medicine (Kalos et al., 2011) for modified T cell immunotherapy in several patients with advanced CLL, the alliance lays groundwork for future pivotal studies that hold potential for expanding use of CAR therapies for additional cancers.
The alliance will include $20 million from Novartis toward construction of a first-of-its-kind Center for Advanced Cellular Therapies on Penn's campus in Philadelphia. The center will be devoted to discovery, development, and manufacturing of adoptive T cell immunotherapies through a joint R&D program led by scientists and clinicians from Penn, Novartis, and the Novartis Institutes for Biomedical Research.
Carl H. June, M.D., director of translational research at Penn's Abramson Cancer Center, told Bloomberg News that Novartis was one of three pharma companies that had negotiated with the university to form the alliance. He would not name the other two. “I never thought this would happen, that the pharma industry would get into ultra-personalized therapy,” added Dr. June, who is also a professor of pathology and laboratory medicine at Penn's Perelman School of Medicine.
In results published in Arteriosclerosis, Thrombosis, and Vascular Biology, a journal published by the American Heart Association, the team cited decreases in the rate of amputation and mortality for up to 2 years after gene therapy compared with historical data (Makino et al., 2012).
During a phase 1/2a investigator-initiated, open-label clinical trial, researchers found increased ankle–brachial pressure index (ABI) in 11 of the 17 patients (65%) at 2 months, and in 11 of 14 patients (79%) at 2 years after therapy. Reduced pain during rest was reported by 8 of 13 patients (62%) at 2 months, and all 9 patients measured at 2 years. The number of ischemic ulcers shrank by more than 25% in 9 of 10 (90%), followed by a reduction in ulcer size, after 2 years.
“Severe complications and adverse effects caused by gene transfer were not detected in any patient throughout the period up to 2 years,” the research team reported.
The study also noted that ABI and maximum walking distance peaked at about 3 to 6 months after therapy, before decreasing slightly, while rest pain and ulcer size improved continuously until 2 years after therapy. A possible explanation for the discrepancy is that improvement in blood flow by HGF gene therapy might increase local flow, resulting in continuous improvement in resting pain and ulcer size, the research team surmised.
Study limitations, the team acknowledged, included it not being randomized or placebo controlled; its small number of patients, because it was a preliminary study before a phase 3 randomized trial; and the dropping out of some patients, which could have resulted in biased long-term results.
“Larger studies to determine whether HGF plasmid gene therapy treatment can avoid major amputation and decrease the mortality in patients with CLI [critical limb ischemia] are warranted,” the team added.
The FDA's approval is the agency's first for any trial focused on treating Cooley's anemia with genetically engineered cells. EGT says its therapy represents a potential cure for a chronic condition, the current treatment for which requires lifelong regular blood transfusions and chelation therapy to allow patients to avoid early death.
The inventor of TNS9.3.55, Michel Sadelain, M.D., Ph.D., is director of the Center for Cell Engineering at the Memorial Sloan-Kettering Cancer Center (MSKCC;
“Our team was the first to show this approach was possible in disease models, and I'm thrilled to be able to finally start offering this potentially curative therapy to patients,” Dr. Sadelain said in an article published at the MSKCC website.
Dr. Sadelain is coleading the trial along with Farid Boulad, M.D., a pediatric hematologist-oncologist and transplant specialist, and Isabelle Rivière, Ph.D., director of the MSKCC Cell Therapy and Cell Engineering Facility.
The trial calls for patients to have their blood stem cells extracted from circulating blood. Investigators will use TNS9.3.55 to introduce a functional version of the β-globin gene into patients' stem cells. After receiving a low dose of chemotherapy to suppress the body's natural production of blood cells, patients will have their own genetically engineered stem cells infused back into them.
Eventually, the trial will be extended to patients at other institutions, including the National Institutes of Health and the University of Washington. Dr. Sadelain's group is coordinating with investigators in Italy and Greece and throughout Asia—areas where β-thalassemia is much more prevalent—to offer the treatment to patients there as well.