Gene editing with adeno-associated virus vector offers hope for hereditary deafness

An R75W mutation in the gap junction β2 (GJB2) gene causes severe fragmentation of gap junction plaques, connecting adjacent cells and leading to syndromic hearing loss. In a new experimental study, scientists from Juntendo University in collaboration with researchers from the University of Tokyo have developed an adeno-associated virus (AAV) vector-mediated genome editing approach to repair the R75W mutation.

Their findings, now published in JCI Insight, could contribute to the development of innovative gene therapies to treat hereditary hearing loss.

Congenital hearing loss refers to impaired auditory function that occurs due to genetic causes. GJB2 is the gene responsible for approximately half of all cases of hereditary hearing loss. Connexin 26 (CX26), which is encoded by GJB2, helps in the formation of intercellular gap junctions—channels that allow for the movement of ions and chemical messenger molecules between adjacent cells, where it regulates auditory function.

GJB2 mutations often lead to fragmentation of gap junctions and gap junction plaques (GJPs) which are composed of CX26. While the inheritance of a recessive GJB2 mutation containing two copies of the defective gene can be functionally cured via GJB2 gene replacement, a GJB2 dominant-negative mutation where the mutant protein inhibits the normal functioning of the wild-type protein necessitates a gene editing approach.

Researchers from Japan have successfully developed a gene therapy to repair R75W, a dominant-negative mutation of GJB2 that causes syndromic hearing loss. The research team included Associate Professor Dr. Kazusaku Kamiya and Assistant Professor Dr. Takao Ukaji in the Department of Otorhinolaryngology, Juntendo University Faculty of Medicine, Japan, and Dr. Osamu Nureki from the Department of Biological Sciences, Graduate School of Science, the University of Tokyo, Japan.

“The overwhelming majority of mutations causing hereditary hearing loss involve the GJB2 gene. However, treatments that can restore hearing in patients with genetic deafness are lacking,” says Dr. Kamiya. “Our research can contribute to the development of gene therapy to tackle the increasing incidence of hereditary hearing loss patients.”

First, the researchers developed an AAV (AAV-Sia6e) that can deliver genome editing tools to a wide range of inner ear cells that form gap junctions. AAV is a useful vector for delivering genes; however, the size of the gene that can be carried is limited.

Therefore, Dr. Kamiya and his team constructed a base editing tool (SaCas9-NNG-ABE8e) that was miniaturized to a size that can be carried by AAV using SaCas9-NNG, which has a smaller size and higher genome editing efficiency than conventional Cas9. Next, they loaded this base editing tool onto AAV-Sia6e, which has high infection tropism for inner ear cells and developed an all-in-one AAV vector for inner ear genome editing.

Genome editing via the all-in-one AAV vector showed considerable efficiency and specificity. It showed on-target T to C conversion in human cells, expressing the GJB2 R75W mutation, repaired the R75W mutation, and formed a clear GJP. Furthermore, after the base editing procedure, the physiological function of gap junction cell-cell communication was restored.

Finally, to validate their results, the researchers performed AAV-mediated genome editing in a transgenic mouse model with the GJB2 R75W mutation. Inner sulcus cells of the cochlea, which were infected with the all-in-one AAV vector, formed distinct GJPs with pentagonal or hexagonal structures arranged around cells that were similar to those observed in wild-type cells.

“By using an all-in-one AAV vector with high infectivity for the inner ear, we expect to improve the therapeutic effect, simplify the development process, and reduce costs. Furthermore, the ABE-based gene editing approach is expected to be less toxic and safer than the conventional CRISPR-Cas9 technology. The AAV genome editing therapy we developed can also be applied to the treatment of other genes that cause hearing loss,” concludes Dr. Kamiya.

Taken together, these findings highlight the immense therapeutic potential of the AAV-mediated genome editing approach for treating hereditary hearing loss.

Provided by
Juntendo University