Patients with Duchenne muscular dystrophy have a mutated gene for dystrophin, a vital muscle cell structural protein. [© artist-unlimited – iStockphoto.com]
Duchenne muscular dystrophy (DMD) and its milder form, Becker muscular dystrophy (BMD), are genetic disorders affecting approximately one in 3,500 new born males. Individuals with DMD have a life expectancy of about 25 years on average, and current estimates place the prevalence of DMD at about 300,000 individuals worldwide.
Patients living with DMD possess a mutated gene for dystrophin, a vital muscle cell structural protein. DMD is described as a recessive X-linked form of muscular dystrophy, which means the disease is carried on the X chromosome and prevalent only in males because men only have one copy of the X chromosome (males are XY) and women have two (XX). Women can be carriers of the disorder but rarely exhibit symptoms of the disease.
Last week, Part I of this two-part series of articles focused on gene editing strategies involving PTC Therapeutics’ small molecule drug candidate PTC124 (ataluren), which acts, the company believes, by interacting with the ribosome. This enables it to read through premature nonsense stop signals on mRNA, thus allowing the cell to produce a full-length, functional protein. The company says that that the small molecule drug has the potential to treat other genetic disorders for which a nonsense mutation is the cause of the disease.
Other tools for targeted DNA cleavage and tailored genome modification are rapidly being developed. In these approaches, the use of sequence-specific DNA binding proteins is being exploited to create new tools for targeted genome editing. Nucleases can be engineered to cleave a desired sequence in the genome to create a double-stranded break. Then the cell’s ability to repair DNA breaks is harnessed to generate a modification of choice at the specific genomic locale. The first class of these engineered molecules, zinc-finger proteins, has entered clinical trials for treatment of diabetic neuropathy, AIDS, and glioblastoma.
Most recently, transcription activator-like effector nucleases (TALENs) have generated a lot of excitement among basic and clinical researchers. These molecules may prove game-changers in correcting mutations that cause severe genetic diseases, allowing researchers to specifically cut any DNA sequence, thereby enabling gene knockouts, knockins, and single base-pair edits.
TALENs comprise a nonspecific DNA-cleaving nuclease fused to a DNA-binding domain that can be easily engineered so that they can target essentially any genomic sequence. The capability to quickly and efficiently alter genes, scientists say, using these molecules promises to have profound impacts on biological research and to yield potential therapeutic strategies for genetic diseases.
As described by scientists who recently reported use of TALENs to repair a gene defect that causes DMD, TALENs are fusion proteins incorporating the catalytic domain of the endonuclease FokI and a designed TALE (transcription activator-like effector) DNA-binding domain. This domain can be targeted to a custom DNA sequence.
The TALE domain consists of an array of repeat variable diresidue modules, each of which specifically recognizes a single base pair of DNA. Repeat variable diresidue modules can be arranged in any order to assemble an array that recognizes a defined sequence.
Site-specific double-strand breaks in DNA are created when two independent TALENs bind to adjacent DNA sequences, thereby permitting dimerization of FokI and cleavage of the target DNA. This targeted double-strand break stimulates cellular DNA repair through either homology-directed repair (HDR) or the nonhomologous end joining (NHEJ) pathway. In this instance, the investigators leveraged the cell’s HDR mechanisms, which use a donor DNA template to guide repair and can be used to create specific sequence changes to the genome, including the targeted addition of whole genes.
Artificial TAL effectors can be designed to bind almost any DNA sequence to enable 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. NHEJ is a pathway that repairs double-strand breaks in DNA.
To date, applications, for TALENs have included, among others, their use to rapidly and efficiently generate mutant alleles of genes in cultured somatic cells or human pluripotent stem cells. Q. Ding and colleagues in the department of stem cell and regenerative biology, Harvard University, reported differentiation of stem cells—both the targeted lines and isogenic control lines into various metabolic cell types. They demonstrate cell-autonomous phenotypes directly linked to disease: dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. Given the speed and ease with which they could derive and characterize these cell lines, the researchers said, they anticipate that TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.
With regard to treating disease, Duke University scientists and colleagues at the Universite Pierre et Marie Curie, Paris, and Universite Laval, Québec, have used TALEN technology to change the existing mutated gene responsible for DMD into a normally functioning dystrophin gene. The Duke researchers believe their approach could be safer and more stable than current methods of gene therapy.
The investigators engineered TALENs to mediate highly efficient gene editing at exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from DMD patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. Exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein-coding regions of the genome, as predicted by in silico target site analysis.
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.