Advance of gene therapy could reverse a g

image: This scanning electron micrograph shows the sensory outer hair cells, which are necessary for cochlear amplification and normal hearing. The image on the left shows a disorganized sensory hair cell in the inner ear of a mouse with a mutation in the Strc gene; as a result, the cell lacks the scaffolding links provided by the stereocilin protein. A neighboring hair cell, on the right, has taken up the double AAV gene therapy vector and has returned to its normal organization. Red arrows indicate the restored stereocilin crosslinks, which erect hair cell microvilli into organized bundles that can contact the overlying tectorial membrane and detect sound vibrations.
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Credit: Carl Nist-Lund, Holt / Géléoc Lab, Boston Children’s Hospital

Hearing loss has been linked to mutations in at least 100 different genes, but up to 16% of genetic hearing loss can be attributed to a single gene, CSTR, the second most common genetic cause. A one-of-a-kind gene therapy technique developed at Boston Children’s Hospital successfully replaced the mutated protein, stereocilin, in the inner ear and reversed severe hearing loss in mice – sometimes at normal hearing levels. The results were published on December 15 in the journal Scientists progress.

The technique could also be used in other situations where the therapeutic gene is very large, says Jeffrey Holt, PhD, a scientist in the departments of otolaryngology and neurology at Boston Children’s and principal investigator of the study.

The team will now test whether the technique works with the human stereocilin gene and test it in human inner ear cells in a dish, derived from patients with CSTR hearing loss. If gene therapy restores hearing function at the tissue level, Holt hopes to seek FDA clearance to test it in humans.

Re-establish contact

In order for sounds to be heard, sensory hair cells in the inner ear must make contact with the tectorial membrane of the ear, which vibrates in response to sound, and then convert those vibrations into signals sent to the brain. The stereocilin protein acts like a scaffold, helping the microvilli of hair cells to come together into an organized bundle, so that their ends can touch the membrane.

“If the stereocilin is mutated, you don’t have that contact, so the hair cells aren’t stimulated properly,” says Holt. “Most importantly, hair cells remain functional, so they are receptive to gene therapy. We believe this will provide a wide window of opportunity for treatment – from babies to hearing-impaired adults. ”

Create a new type of gene therapy

To deliver a healthy stereocilin gene, the team used a synthetic adeno-associated virus (AAV) effective to target hair cells.

“The challenge we faced was that the stereocilin gene is too big to fit in the gene therapy vector,” says Holt. “The gene is about 6,200 base pairs of DNA in length, but AAV only has a capacity of 4,700 base pairs.”

Olga Shubina-Oleinik, PhD, the study’s first author, came up with a solution: she split the mouse Strc gene in two, and put the two halves in two separate AAVs. She then used an existing technique called protein recombination, where two halves of a protein lie and connect. But in this case, it didn’t work.

“We then realized that the beginning of the protein has a short stretch of amino acids that acts as an ‘address’, directing the protein to its proper place in the cell,” explains Shubina-Oleinik. “When we split the protein in half, we realized that one half had the signal, but the other didn’t, so the halves might not end up in the same place.”

When they added the signal to the two halves of the protein, the two halves successfully joined together. The researchers found a robust restoration of the full-length stereocilin protein in mice and normal-looking hair bundles that may have come in contact with the tectorial membrane.

Hearing restoration

The researchers used two types of hearing tests: one similar to hearing tests used in babies, and a test that uses electrodes on the scalp to measure the brainstem’s auditory responses to a range of frequencies and sound intensities. In testing, the mice were found to be much more sensitive to subtle sounds and showed improved cochlear amplification – the ability to amplify weak sounds, dampen the response to loud sounds, and more accurately discriminate between sounds of different frequencies. . In some mice, hearing was restored to normal levels.

“The results were remarkable and are the first example of hearing restoration using dual vector gene therapy to target sensory outer hair cells,” explains Shubina-Oleinik.

Shubina-Oleinik and Holt have applied for a patent for gene therapy technology. According to co-author Eliot Shearer, MD, PhD, clinician-researcher in otolaryngology at Boston Children’s Hospital, approximately 100,000 patients in the United States and 2.3 million worldwide carry CSTR mutations and could potentially benefit from this therapy.

“Actually CSTR genetic variations are more common than we thought, which makes gene therapy for this disorder so important, ”says Shearer, who worked with the Children’s Rare Disease Cohort Initiative to screen a large genomic dataset for CSTR mutations.

The study was supported by the IDDRC (Grant # 1U54HD090255), the Boston Children’s Hospital Rare Disease Cohort Initiative, the Jeffrey and Kimberly Barber Fund for Gene Therapy Research, the Usher Syndrome Society, and the Foundation Pour L’Audition . Holt is a scientific founder of Audition Therapeutics and an advisor to several biotechnology companies focused on inner ear therapy, and is the inventor of a patent for the use of AAV9-PHP.B for delivery of genes in the inner ear. The authors declare no other competing interests.

About Boston Children’s Hospital

Boston Children’s Hospital is classified on # 1 children’s hospital in the nation by US News & World Report and is the primary pediatric education affiliate of Harvard Medical School. Home to the world’s largest pediatric medical center-based research company, its findings have benefited both children and adults since 1869. Today, 3,000 researchers and scientific staff, including 10 members of the National Academy of Sciences, 25 members of the National Academy of Medicine and 10 Howard Hughes medical researchers form the Boston Children’s research community. Founded as a 20-bed children’s hospital, Boston Children’s is now a comprehensive 415-bed center for pediatric and adolescent health care. To learn more, visit our Answers blog and follow us on social media @BostonChildrens, @BCH_Innovation, Facebook and Youtube.

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