AAV-Mediated Gene Therapy Restores Hearing in Patients with DFNB9 Deafness

Abstract

Mutations in OTOFERLIN (OTOF) lead to the autosomal recessive deafness 9 (DFNB9). The efficacy of adeno-associated virus (AAV)-mediated OTOF gene replacement therapy is extensively validated in Otof-deficient mice. However, the clinical safety and efficacy of AAV-OTOF is not reported. Here, AAV-OTOF is generated using good manufacturing practice and validated its efficacy and safety in mouse and non-human primates in order to determine the optimal injection dose, volume, and administration route for clinical trials. Subsequently, AAV-OTOF is delivered into one cochlea of a 5-year-old deaf patient and into the bilateral cochleae of an 8-year-old deaf patient with OTOF mutations. Obvious hearing improvement is detected by the auditory brainstem response (ABR) and the pure-tone audiometry (PTA) in these two patients. Hearing in the injected ear of the 5-year-old patient can be restored to the normal range at 1 month after AAV-OTOF injection, while the 8-year-old patient can hear the conversational sounds. Most importantly, the 5-year-old patient can hear and recognize speech only through the AAV-OTOF-injected ear. This study is the first to demonstrate the safety and efficacy of AAV-OTOF in patients, expands and optimizes current OTOF-related gene therapy and provides valuable information for further application of gene therapies for deafness.

Keywords: AAV; OTOF gene therapy; biosafety; clinical trial; hearing recovery.

Figure 1

Experimental design. A) Human OTOF expression method. Full‐length OTOF was packaged into dual AAV vectors and acquired by DNA recombination via the AK sequence and RNA trans‐splicing via the splicing donor (SD) and splicing acceptor (SA) after delivery. OTOF expression was driven by the mouse hair cell‐specific promoter. The therapeutic AAVs were manufactured in accordance with the GMP level. B) The experimental and therapeutic design of AAV‐OTOF therapy in mice, cynomolgus macaques, and patients with OTOF mutations.

Figure 2

AAV‐OTOF delivery efficiently restored hearing in adult OTOF^Q939*/Q939* mice. A) The click ABR results in OTOF^Q939*/Q939* mice, wild type (WT) mice, and the P30 OTOF^Q939*/Q939* mice injected with AAV‐OTOF after 1 week and 1 month, respectively. B) The ABR results from OTOF^Q939*/Q939* mice, WT mice, and the P30 OTOF^Q939*/Q939* mice injected with AAV‐OTOF after 1 week and 1 month, respectively. C) The ABR results of WT mice, all OTOF^Q939*/Q939* mice, and the best performing OTOF^Q939*/Q939* mice injected with AAV‐OTOF after 1 week and 1 month, respectively. D) The confocal images of OTOF‐N and OTOF‐C in adult OTOF^Q939*/Q939* mice injected with AAV‐OTOF after 7 days. OTOF‐N: red, OTOF‐C: cyan, phalloidin: blue. Scale bar: 50 µm. E) The percentage of OTOF‐positive IHCs in (D). All data are shown as the mean ± SEM. The p‐value was calculated by Student's t‐test. ***p < 0.001 and ****p < 0.0001.

Figure 3

AAV‐OTOF was audiologically and systemically safe in cynomolgus macaques. A) The experimental procedure in cynomolgus macaques. B) The images of round window membrane exposure and AAV injection in cynomolgus macaques. C) The ABR results of click and tone‐burst in cynomolgus macaques injected with menstruum, low dose AAV‐OTOF, and high dose AAV‐OTOF, respectively. I: ipsilateral ear, C: contralateral ear. D) The HE staining of tissues from control and AAV‐OTOF injected cynomolgus macaques. Scale bar: 100 µm.

Figure 4

AAV‐OTOF was delivered into the cochlea through the trans‐mastoid facial recess. A) The experimental procedure in the patient. B) The images show the exposure of the round window membrane and AAV injection in the patient. The exposure of the mastoid and round window was performed under a surgical microscope, and injection of AAV‐OTOF was administrated via an oto‐endoscope.

Figure 5

AAV‐OTOF rescued hearing in the 5‐year‐old patient with OTOF mutations. A) Genomic DNA copies of AAV‐OTOF‐N and AAV‐OTOF‐C and the NAbs levels from the patient at 1 day (D1), 3 days (D3), 1 week (W1), 2 weeks (W2), 3 weeks (W3), 1 month (M1) and 2 months (M2) after AAV injection, respectively. The arrows indicate that the DNA copies were below the quantification limit (BQL). B,C) Representative traces recorded for the click ABR from the patient at 2 weeks and 1 month after AAV‐OTOF injection. The recording was made twice at each sound intensity. Wave V is indicated by the arrows. D) The click ABR results of the patient. E) Representative traces recorded for tone‐burst ABR at 2 kHz and 4 kHz from the patient at 1 month after AAV‐OTOF injection. The recording was made twice at each sound intensity. Wave V is indicated by the arrows. F) The tone‐burst ABR results of the patient. G) The PTA results of the patient after AAV‐OTOF injection. And the PTA thresholds of the contralateral ears with the CI turning on were also shown. The arrows in (D, F, G) indicate that the patient had no responses to the maximum sound intensity at each frequency.

Figure 6

AAV‐OTOF rescued hearing in the 8‐year‐old patient with OTOF mutations. A) Genomic DNA copies of AAV‐OTOF‐N and AAV‐OTOF‐C and the NAbs levels from the patient at 1 day (D1), 3 days (D3), 1 week (W1), 2 weeks (W2) and 1 month (M1) after AAV injection, respectively. The arrows indicate that the DNA copies were below the quantification limit (BQL). B) Representative traces recorded for tone‐burst ABR at 2 kHz at 2 months after AAV‐OTOF injection. The recording was made twice at each sound intensity. Wave V is indicated by the arrows. C) The click ABR results of the patient. D) Representative traces recorded for tone‐burst ABR at 2 kHz of the right ear from the patient at 30/40 days after AAV‐OTOF injection. The recording was made twice at each sound intensity. Wave V is indicated by the arrows. E) The tone‐burst ABR results of right ear from the patient. F,G) Similar results as (D‐E) excepted that the left ear was analyzed. H) The PTA results of the patient before and after AAV‐OTOF injection. The arrows in (C, E, G, H) indicate that the patient had no responses to the maximum sound intensity at each frequency.