Researchers at Harvard Medical School and Boston Children's Hospital used an optimized gene-editing system to recover the hearing of mice with genetic hearing loss. They were successful in doing so without any unwanted side effects from the treatment. The new approach involves a more precise and enhanced version of the CRISPR-Cas9 gene-editing system, which is better at recognizing the mutation that causes progressive hearing loss in the animal models known as Beethoven mice. This tool gave the researchers the ability to disable the defective copy of a hearing gene called Tmc1 without disturbing the healthy copy.
“The most surprising aspect was that it worked so well, giving almost undetectable cutting of inappropriate genes,” said study co-senior investigator David Corey, PhD. “This greatly reduces risk in a clinical setting, and means that CRISPR approaches may be used for gene therapy sooner.”
The Beethoven mice were treated for the same genetic mutation that causes progressive hearing loss in humans. Without the therapy, the mice are completely deaf after reaching six months of age. With the therapy, recently described in Nature Medicine, the mice can detect sounds at about 45 dB, the level of a typical conversation. In the mice, the mutation is marked by one incorrect letter, an A instead of a T, in the DNA sequence of the Tmc1 gene. This single incorrect letter is the difference between normal hearing and deafness.
The system managed to recognize a single incorrect DNA letter in a defective copy among 3 billion letters in the mice genome. Despite this success, much work is needed before the gene-editing therapy can be used on humans, according to the study authors. The new therapy, however, is necessary for improving the safety and effectiveness of the standard gene-editing technique.
“The particular mutation that we studied in mice is rather rare in humans, somewhat limiting the impact of the study,” Corey told The Hearing Journal. “But when we analyzed the DNA sequences for known disease-causing mutations in thousands of other genes, we were surprised that a great many, almost a quarter, could be targeted with this same modified SaCas9-KKH.”
The classic CRISPR-Cas9 gene-editing tools work by using a guiding molecule, gRNA, to identify the mutant DNA sequence. Once the mutant DNA is found, the Cas9 enzyme will cut it off. Until this point, gene editors haven't been the most accurate. To surpass this challenge, the researchers adapted a tool, originally developed by Keith Joung, MD, PhD, and Ben Kleinstiver, PhD, using a modified Cas9 enzyme from Staphylococcus aureus instead of the bacterium Streptococcus pyogenes, from which the standard Cas9 is derived. To enhance the detection accuracy, the new system uses the gRNA to locate the mutant gene and an enhanced Cas9 to locate the specific DNA mutation.
“The critical idea in this study was the use, in addition to the guide RNA, of the ‘PAM site’ in the modified SaCas9-KKH enzyme to mediate a secondary recognition of the mutant gene,” said Corey. “We hoped that this would reduce the off-target effects of the Cas9, either inappropriate cutting of the normal copy of the TMC1 gene or inappropriate cutting of other unrelated genes.”
Corey and his team then injected the treatment into the mice's inner ears. Further analysis showed that the tool worked since the editing only occurred in the inner ear of the mice. To analyze this evidence, the researchers stimulated the hair cells of the mice that didn't carry the mutant cell. They also measured the mice's auditory brainstem responses to determine if the therapy worked.
Because of the tool's ability to target single-point gene mutations, the system has the potential to mediate 15 other forms of inherited deafness also caused by a single-letter mutation in the DNA sequence of other hearing genes.
“We will work with colleagues in the field of hearing and deafness to apply this technique to other forms of hereditary deafness,” said Corey. “We hope that others in the gene therapy community will pick this up to work toward the treatment of other disorders.