Targeted to Novel Sequences
Artificial TAL effectors targeted to novel sequences can activate transcription, thus enabling multiple TAL effector-based genome engineering applications. Since their discovery, sequence-specific DNA-binding proteins with predicted binding specificities have been generated economically in a matter of days, using molecular biology methods practiced by most laboratories. The activities of custom-designed TALENs in human cells have efficiencies of NHEJ-induced mutagenesis ranging up to 45% of transfected cells.
“Conventional genetic approaches to treating the disease involve adding normal genes to compensate for the mutated genes,” said Charles Gersbach, Ph.D., assistant professor of biomedical engineering at Duke's Pratt School of Engineering and the department of orthopedic surgery. “However, this can cause other unforeseen problems, or the beneficial effect does not always last very long.”
“Our approach actually repairs the faulty gene, which is a lot simpler,” said David Ousterout, the Duke biomedical engineering graduate student in the Gersbach lab who led the work. “It finds the faulty gene, and fixes it so it can start producing a functional protein again.”
The scientists say that this strategy integrates the rapid and robust assembly of active TALENs with an efficient gene-editing method for the correction of genetic diseases caused by mutations in nonessential coding regions that cause frameshifts or premature stop codons.
Potentially, this approach could treat more than 60% of DMD patients, who have a variety of mutations in the dystrophin gene. Dr. Gersbach explained to GEN, “Because it’s specific to the gene sequence, you would have to customize the enzyme to the type of mutation. The one that we made would address about 15% of patients. But you could make others that would work on nearby sequences and fix the entire gene.”
Dr. Gersbach says his group is now conducting further tests of this new approach in animal models of the disease, as well as exploring delivery of the gene correcting enzymes directly to muscles.
And the researchers say, similar approaches using TALEN technology could help treat other genetic diseases where a few gene mutations are responsive, such as sickle cell diseases, hemophilia, or other muscular dystrophies.
Several companies, including Transposagen, Cellectis, and Life Technologies, supply these site-specific nucleases for DNA editing. A plasmid kit for assembling custom TALEN and other TAL effector constructs is available through the public, not-for-profit repository Addgene.