Articles
www.thelancet.com Published online January 24, 2024 https://doi.org/10.1016/S0140-6736(23)02874-X 1
AAV1-hOTOF gene therapy for autosomal recessive
deafness 9: a single-arm trial
Jun Lv*, Hui Wang*, Xiaoting Cheng*, Yuxin Chen*, Daqi Wang*, Longlong Zhang, Qi Cao, Honghai Tang, Shaowei Hu, Kaiyu Gao, Mengzhao Xun,
Jinghan Wang, Zijing Wang, Biyun Zhu, Chong Cui, Ziwen Gao, Luo Guo, Sha Yu, Luoying Jiang, Yanbo Yin, Jiajia Zhang, Bing Chen,
Wuqing Wang†, Renjie Chai†, Zheng-Yi Chen†, Huawei Li†, and Yilai Shu†
Summary
Background Autosomal recessive deafness 9, caused by mutations of the OTOF gene, is characterised by congenital or
prelingual, severe-to-complete, bilateral hearing loss. However, no pharmacological treatment is currently available
for congenital deafness. In this Article, we report the safety and efficacy of gene therapy with an adeno-associated
virus (AAV) serotype 1 carrying a human OTOF transgene (AAV1-hOTOF) as a treatment for children with autosomal
recessive deafness 9.
Methods This single-arm, single-centre trial enrolled children (aged 1–18 years) with severe-to-complete hearing loss
and confirmed mutations in both alleles of OTOF, and without bilateral cochlear implants. A single injection of
AAV1-hOTOF was administered into the cochlea through the round window. The primary endpoint was dose-limiting
toxicity at 6 weeks after injection. Auditory function and speech were assessed by appropriate auditory perception
evaluation tools. All analyses were done according to the intention-to-treat principle. This trial is registered with
Chinese Clinical Trial Registry, ChiCTR2200063181, and is ongoing.
Findings Between Oct 19, 2022, and June 9, 2023, we screened 425 participants for eligibility and enrolled six children
for AAV1-hOTOF gene therapy (one received a dose of 9 × 10¹¹ vector genomes [vg] and five received 1·5 × 10¹² vg). All
participants completed follow-up visits up to week 26. No dose-limiting toxicity or serious adverse events occurred. In
total, 48 adverse events were observed; 46 (96%) were grade 1–2 and two (4%) were grade 3 (decreased neutrophil
count in one participant). Five children had hearing recovery, shown by a 40–57 dB reduction in the average auditory
brainstem response (ABR) thresholds at 0·5–4·0 kHz. In the participant who received the 9 × 10¹¹ vg dose, the average
ABR threshold was improved from greater than 95 dB at baseline to 68 dB at 4 weeks, 53 dB at 13 weeks, and 45 dB at
26 weeks. In those who received 1·5 × 10¹² AAV1-hOTOF, the average ABR thresholds changed from greater than
95 dB at baseline to 48 dB, 38 dB, 40 dB, and 55 dB in four children with hearing recovery at 26 weeks. Speech
perception was improved in participants who had hearing recovery.
Interpretation AAV1-hOTOF gene therapy is safe and efficacious as a novel treatment for children with autosomal
recessive deafness 9.
Funding National Natural Science Foundation of China, National Key R&D Program of China, Science and Technology
Commission of Shanghai Municipality, and Shanghai Refreshgene Therapeutics.
Copyright © 2024 Elsevier Ltd. All rights reserved.
Introduction
Up to 60% of cases of congenital deafness, which affects
approximately 26 million people worldwide, are caused
by genetic mutations.1,2 Autosomal recessive deafness 9,
characterised by severe-to-complete, congenital or
prelingual, bilateral hearing loss, results from
dysfunction of otoferlin (encoded by the OTOF gene)3
and accounts for 2–8% of cases of congenital deafness.4–7
Autosomal recessive deafness 9 has profound effects on
speech development if not treated early in life.8,9
Gene therapy has previously shown success in treating
various human diseases caused by mutation of a single
gene,10,11 and studies in animal models have established
the efficacy of gene therapy for congenital hearing loss.12
However, the safety and efficacy of gene therapy on
congenital hearing loss in humans is poorly explored.
We and other groups have reported restoration of
auditory function in Otof (knockout) mouse models via
gene replacement with the otoferlin coding sequence
delivered by dual-adeno-associated virus (AAV) vectors,
which permits circumvention of the size limitation
of a single AAV (which cannot accommodate a full-
length otoferlin coding sequence).13–15 We subsequently
designed the AAV1-hOTOF vector-based gene therapy
carry ing the human otoferlin coding sequence driven by
Myo15, a hair cell-specific promoter, and verified its
efficacy and safety in mice and the safety of AAV1 vector-
carrying MYO15 and a reporter transgene in non-human
primates.16 Here, we report the results of a single-arm
trial in which we investigated the safety and efficacy of
AAV1-hOTOF treatment in children with autosomal
recessive deafness 9.
Published Online
January 24, 2024
https://doi.org/10.1016/
S0140-6736(23)02874-X
*Joint first authors
†Joint last authors
ENT Institute and
Otorhinolaryngology
Department, Eye & ENT
Hospital (J Lv MMed,
H Wang MD, X Cheng MD,
Y Chen PhD, D Wang PhD,
L Zhang MMed, Q Cao BMed,
H Tang PhD, S Hu PhD,
M Xun BMed, J Wang MD,
Z Wang BMed, B Zhu PhD,
C Cui BMed, Z Gao PhD,
L Guo PhD, S Yu MD,
L Jiang BMed, Y Yin MMed,
J Zhang MMed, Prof B Chen MD,
Prof W Wang MD, Prof H Li MD,
Prof Y Shu MD), NHC Key
Laboratory of Hearing
Medicine (J Lv, H Wang, X Cheng,
Y Chen, D Wang, L Zhang, Q Cao,
H Tang, S Hu, M Xun, J Wang,
Z Wang, B Zhu, C Cui, Z Gao,
L Guo, S Yu, L Jiang, Y Yin,
J Zhang, Prof B Chen,
Prof W Wang, Prof H Li,
Prof Y Shu), State Key
Laboratory of Medical
Neurobiology and MOE
Frontiers Center for Brain
Science (J Lv, C Cui, L Jiang,
J Zhang, Prof H Li, Prof Y Shu),
Institutes of Biomedical
Science (J Lv, C Cui, L Jiang,
J Zhang, Prof H Li, Prof Y Shu),
Fudan University, Shanghai,
China; Research and
Development Department,
Shanghai Refreshgene
Therapeutics, Shanghai, China
(K Gao PhD); State Key
Laboratory of Digital Medical
Engineering (Prof R Chai DPhil),
Department of Otolaryngology
Head and Neck Surgery of
Zhongda Hospital (Prof R Chai),
Advanced Institute for Life and
Health (Prof R Chai), Jiangsu
Province High-Tech Key
Laboratory for Bio-Medical
Research (Prof R Chai),
Southeast University, Nanjing,
China; Co-Innovation Center of
Neuroregeneration, Nantong
University, Nantong, China
Articles
2 www.thelancet.com Published online January 24, 2024 https://doi.org/10.1016/S0140-6736(23)02874-X
Methods
Study design and participants
This single-arm trial was done at the Eye & ENT Hospital
of Fudan University (Shanghai, China). Children (aged
1–18 years) of either sex were eligible if they had
autosomal recessive deafness 9 due to biallelic pathogenic
(or likely pathogenic) OTOF mutations and severe-to-
complete hearing loss, as defined by average auditory
brainstem response (ABR) threshold (0·5, 1·0, 2·0, and
4·0 kHz) at 65 dB or greater.17 Before each hearing test,
the participant’s body temperature was measured to
confirm that it was within a normal range of 36–37°C.
Participants with bilateral cochlear implants were
excluded. Blood samples were obtained from the
participants and both their biological parents. The
genotype of participants was assessed by whole-exome
sequencing and OTOF variants in participants and their
biological parents were also detected by Sanger
sequencing. The pathogenicity of variants was confirmed
by agreement from three independent geneticists (LG
and SY plus another geneticist not otherwise affiliated
with the trial) according to the latest version of the
American College of Medical Genetics and Genomics’
and Association for Molecular Pathology’s Variant
Interpretation Guidelines and ClinGen Hearing Loss
Expert Panel Specifications. For safety reasons, the first
three participants were required to be at least 3 years old;
subsequent participants could be enrolled from the age
of 1 year. Participants were excluded if they produced
AAV1-neutralising antibodies at a titre of 1:2000 or
greater. Detailed inclusion and exclusion criteria are
listed in the protocol (appendix pp 76–78).
This study was done in accordance with the Declaration
of Helsinki and Good Clinical Practice guidelines. The
trial protocol ([2022]2022085-1) was approved by the
Ethics Committee of the Eye & ENT Hospital of Fudan
University in June, 2022, and subsequent amendments
were approved by the same committee. Written informed
consent was obtained from the legal guardians (both
parents) of the children before any protocol-related
procedure commenced.
Procedures
AAV1-hOTOF was developed by researchers at the Eye &
ENT Hospital of Fudan University and Refreshgene
Therapeutics (Shanghai, China), and manufactured by
PackGene Biotechnology (Guangzhou, China). For each
participant, a single, minimally invasive injection of
AAV1-hOTOF was administered to one ear through the
round window membrane with stapes fenestration, on
the side with the most severe hearing loss or, for
participants with a cochlear implant, on the side with no
cochlear implant. The planned escalating doses were
30 μL (9 × 10¹¹ vector genomes [vg]) per ear or
50 μL (1·5 × 10¹² vg) per ear; all doses contained a 1:1
mixture of AAV1-hOTOF NT (the 5’ terminal segment of
the OTOF coding sequence) and AAV1-hOTOF CT (the
3ʹ terminal segment of the OTOF coding sequence).
Participants were enrolled sequentially after a dose-
limiting toxicity assessment was completed within
6 weeks for the first participant at each dose group
(appendix p 10). Details of screening, enrolment, and
surgical procedures are described in the appendix (pp 4,
10–11).
To minimise the risk of a potential inflammatory
response, dexamethasone was given intravenously at
0·3 mg/kg per day for 8 consecutive days, starting 3 days
before the AAV1-hOTOF injection. To minimise the risk
of infection, ceftriaxone was given intravenously at
80 mg/kg per day for 5 consecutive days, starting on the
day of AAV1-hOTOF injection. Either CT or MRI was
done at baseline and at 6 weeks to investigate the
structure of ear. At baseline, 3 days, 7 days, 2 weeks,
4 weeks, 6 weeks, 13 weeks, and 26 weeks, urine samples
(Prof R Chai); Department of
Neurology of Aerospace Center
Hospital (Prof R Chai), School of
Life Science (Prof R Chai),
Beijing Institute of Technology,
Beijing, China; Department of
Otolaryngology-Head and
Neck Surgery
(Prof Z Chen DPhil), Graduate
Program in Speech and Hearing
Bioscience and Technology and
Program in Neuroscience
(Prof Z Chen), Harvard Medical
School, Boston, MA, USA;
Eaton-Peabody Laboratory,
Massachusetts Eye and Ear,
Boston, MA, USA (Prof Z Chen)
Correspondence to:
Yilai Shu, ENT Institute and
Otorhinolaryngology
Department, Eye & ENT Hospital
of Fudan University,
Shanghai, 200031, China
yilai_shu@fudan.edu.cn
Research in context
Evidence before this study
We searched PubMed from inception to Oct 1, 2023, for all studies
in English on OTOF mutations, their associations with congenital
hearing loss, including autosomal recessive deafness 9 (DFNB9),
and all related animal preclinical and human clinical trials. The
search terms included “OTOF”, “DFNB9”, “hereditary hearing loss”,
“gene therapy”, “DFNB9 trial”, “DFNB9 mouse”, or combinations
thereof. We also searched ClinicalTrials.gov for related clinical
trials. We found proof-of-principle of gene therapy for DFNB9 in
animal models using recombinant adeno-associated viral vectors
and four clinical trials. We found no reports on the safety or
efficacy of human gene therapy to treat DFNB9.
Added value of this study
To our knowledge, this study is the first prospectively registered
and the first-in-human clinical trial with the largest number of
patients and the longest follow-up published to date of gene
therapy targeting OTOF to treat autosomal recessive
deafness 9. These data indicate that adeno-associated virus
(AAV) administration in the human inner ear is safe and
efficacious in treating genetic hearing loss. The study extends
the utility of dual AAV to overcome the gene size limit to treat
human diseases.
Implications of all the available evidence
Our study provides evidence of the safety and efficacy of gene
therapy to treat autosomal recessive deafness 9 and lays a
foundation for gene therapy as a novel treatment for other
forms of genetic hearing loss. The process and techniques
developed in this study are likely to advance the field of gene
therapy for hearing loss.
See Online for appendix
Articles
www.thelancet.com Published online January 24, 2024 https://doi.org/10.1016/S0140-6736(23)02874-X 3
were collected for routine urine tests and blood samples
were collected for routine blood tests and for blood
biochemistry, coagulation function tests, AAV1-neu-
tralising antibodies tests, interferon-gamma enzyme-
linked immunosorbent spot assays, or vector DNA in
circulation. AAV1-neutralising antibodies and interferon-
gamma were assessed at baseline, 6 weeks, and 13 weeks,
and circulating vector DNA was assessed at baseline and
at 1 week.
Outcomes
The primary endpoint was dose-limiting toxicity, defined
as haematologic toxicity of grade 4 or worse, non-
haematologic toxicity of grade 3 or worse, or aural
toxicity of grade 2 or worse within 6 weeks of injection,
graded according to Common Terminology Criteria for
Adverse Events (version 5.0). Secondary outcomes were
preliminary efficacy (ie, auditory function and speech
perception) and safety. Auditory function was assessed
using ABR, auditory steady-state response, pure-tone
audiometry, and distortion product otoacoustic emission
test at baseline and at 4, 6, 13, and 26 weeks. The average
thresholds of ABR, auditory steady-state response, or
pure-tone audiometry were calculated as the arithmetic
average thresholds at 0·5, 1, 2, and 4 kHz.17 Addtionally,
questionnaires were used to assess auditory function
and speech perception: the Meaningful Auditory
Integration Scale,18 the Infant-Toddler Meaningful
Auditory Inte gration Scale,18 the Categories of Auditory
Performance score,19 the Speech Intelligibility Rating
score,20 and Meaningful Use of Speech Scale.21 Speech
perception was also assessed by Mandarin Speech
Perception software (version 5.04.01)22 and the Angel
Test software (version 5.01.01).23,24 Hearing recovery was
defined as a 10 dB reduction in the average ABR
threshold, as adopted from current guidelines for
sudden sensorineural hearing loss.25 Video head impulse
test was used to assess vestibular function at baseline, 4,
6, 13, and 26 weeks. Safety was measured by the presence
of adverse events, defined as any unfavourable and
unintended sign (including an abnormal laboratory
finding), symptom, or disease temporally associated
with the use of a medical treatment or procedure that
might or might not be considered related to the medical
treatment or procedure. Adverse events will be recorded
after treatment until the trial is complete at 52 weeks.
Details of outcomes are described in the appendix
(pp 5–9). Otoscopic examination was done at weeks 13
and 26 to confirm healing of the tympanic membrane
after the injection.
Statistical analysis
All analyses were based on the intention-to-treat principle.
All analyses, including demographic characteristics,
safety, auditory function, and speech recognition, were
descriptively summarised. This trial is registered at the
Chinese Clinical Trial Registry, ChiCTR2200063181, and
is ongoing.
Role of the funding source
The commercial funder of the study was involved in
study design, protocol amendment, data analysis,
interpretation of data, manuscript revisions, and decision
for submission. All other funding sources had no role in
study design, data collection, data analysis, data
interpretation, the writing of the report, or the decision
to submit the paper for publication.
Results
Between Oct 19, 2022, and June 9, 2023, we screened
425 participants for eligibility and enrolled six eligible
participants (appendix p 10). One participant received an
Participant 1 Participant 2 Participant 3 Participant 4 Participant 5 Participant 6
Sex Female Male Female Male Female Male
Age, years 4·8 5·0 6·2 2·1 3·3 1·0
Ethnicity Han Han Han Han Han Han
OTOF (HGNC:8515) mutations
Mutation in allele 1 c.2985C>A
(p.Cys995*)
c.2215-1G>C c.4961-2A>C c.2215-1G>C c.3409-11A>G c.5647C>T
(p.Gln1883*)
Mutation in allele 2 c.5203C>T
(p.Arg1735Trp)
c.5108delinsTCTT
(p.Arg1703delinsLeuPhe)
c.5567G>A
(p.Arg1856Gln)
c.4225A>T
(p.Lys1409*)
c.5647C>T
(p.Gln1883*)
c.5728G>A
(p.Glu1910Lys)
Hearing threshold†
Auditory brainstem response, dB >95‡ >95 >95 >95 >95 >95
Auditory steady-state response, dB 80 111 98 100 >98 100
Pure-tone audiometry, dB >115 100 106 NA§ NA§ NA§
Cochlear implant Right ear Left ear Right ear None Right ear None
Vector dose administered, vg 9 × 10¹¹ 1·5 × 10¹² 1·5 × 10¹² 1·5 × 10¹² 1·5 × 10¹² 1·5 × 10¹²
NA=not available. vg=vector genomes. *Nonsense mutation. †Average hearing threshold at 0·5–4·0 kHz; the symbol “>” in hearing threshold means no response at
maximum sound intensity level. ‡Only click-evoked auditory brainstem response was tested at baseline in participant 1; at baseline, auditory brainstem response was
measured at 0·25, 0·50, 1·00, 2·00, and 4·00 kHz in the other five participants. §Participants 4, 5, and 6 could not complete pure-tone audiometry due to their young age.
Table 1: Baseline characteristics, genotype, and vector dose for each participant
Articles
4 www.thelancet.com Published online January 24, 2024 https://doi.org/10.1016/S0140-6736(23)02874-X
AAV1-hOTOF dose of 9 × 10¹¹ vg and five participants
received AAV1-hOTOF doses of 1·5 × 10¹² vg. Median
follow-up was 26 weeks (IQR 26–26) and all six participants
completed the 26-week assessment. The median age of
participants was 4·1 years (IQR 2·4–5·0), three were
girls, three were boys, and all were of Han ethnicity
(table 1). Participants 1, 2, 3, and 5 had a unilateral
cochlear implant, and participants 4 and 6 had no cochlear
implant. All identified variants in the OTOF gene were
classified as pathogenic or likely pathogenic and all
enrolled participants had complete hearing loss (no ABR
response at a stimulus of 95 dB) at baseline (table 1).
In the participant who received the 9 × 10¹¹ vg
AAV1-hOTOF dose (participant 1), six adverse events (all
grade 1–2) were observed within the 26-week follow-up
(table 2). In the five participants who received 1·5 × 10¹²
vg AAV1-hOTOF, 42 adverse events were observed
(table 2); all were grade 1–2, except for two grade 3 events
of decreased neutrophil count in participant 5 (appendix
p 15), which resolved spontaneously. No dose-limiting
toxicity or serious adverse event was observed.
Two participants (4 and 5) in the 1·5 × 10¹² vg group had
three events of slightly prolonged activated partial
thromboplastin time (<1·2 times the upper limit of
normal [ULN] range; appendix p 15). Three participants
(4, 5, and 6) had four events of transient reduction in
fibrinogen, but no signs of haemorrhage. Increased
lactate dehydrogenase (<1·5 times higher than the ULN
range; appendix p 15) was observed in five participants
(all but participant 2), without clinical manifestations.
We found no increases in alanine aminotransferase or
serum bilirubin concentrations in any participants.
Aspartate aminotransferase (reference range 15–37 U/L)
increased in participant 1 (39 U/L at 1 week after injection)
and participant 5 (39 U/L at 2 weeks after injection);
neither participant reached Hy’s law criteria for
liver injury, and both cases of increased aspartate
aminotransferase resolved spontaneously by 4 weeks
after injection (appendix p 15). Vestibular function was
normal in participants 1, 2, 3, and 5 at baseline and at 4,
6, 13, and 26 weeks after injection (appendix p 12);
participants 4 and 6 could not finish the test because of
their young age. Otoscopic examination showed healing
of the tympanic membrane (appendix p 13); all
participants had an otoscopic examination at 26 weeks,
except participant 6, who had an examination at 13 weeks.
No obvious ear abnormalities were observed by
radiographic assessments in any participant (data not
shown).
All participants had an increase in AAV1-neutralising
antibodies from baseline to weeks 6 and 13 (table 3).
T cell responses to the AAV1 capsid, as reflected by the
concentration of interferon gamma, were negative for all
participants at both 6 weeks and 13 weeks (table 3).
Vector DNA in the blood was not detectable in any
participant at 7 days (table 3).
In the participant injected with the 9 × 10¹¹ vg dose
(participant 1), the click-evoked ABR threshold was
greater than 95 dB at baseline, and the average ABR
threshold was 68 dB at 4 weeks, 70 dB at 6 weeks, 53 dB
at 13 weeks, and 45 dB at 26 weeks (figure A; appendix
p 16). The best recovery of the ABR threshold was 35 dB
at 0·25 kHz and 2 kHz at 26 weeks (appendix p 16). The
average auditory steady-state response threshold was
80 dB at 4 weeks, 73 dB at 6 weeks, 60 dB at 13 weeks, and
38 dB at 26 weeks (figure A). The average pure-tone
audiometry threshold was 71 dB at 4 weeks, 68 dB at
6 weeks, 55 dB at 13 weeks, and 30 dB at 26 weeks. The
signal-to-noise ratio of the distortion product otoacoustic
emission test was slightly lower at 4 weeks compared
with the baseline, but gradually recovered within
26 weeks (appendix p 14). The noise floor of the distortion
product otoacoustic emission test results is provided in
the appendix (p 17).
In the 1·5 × 10¹² vg group, one participant (participant 2)
did not show hearing improvement within the 26-week
follow-up (figure B; appendix p 16). We found robust
hearing improvement in participants 3, 4, 5, and 6
(figure C–F; appendix p 16). In participant 3, the average
ABR threshold was greater than 95 dB at baseline, 60 dB
at 4 weeks, 63 dB at 6 weeks, 63 dB at 13 weeks, and
48 dB at 26 weeks (figure C). The average auditory
steady-state response was gradually reduced to 55 dB at
26 weeks from 98 dB at baseline, and the average pure-
tone audiometry was gradually reduced to 45 dB at
26 weeks from 106 dB at baseline (figure C). In
participant 4, the average ABR threshold was reduced
from greater than 95 dB at baseline to 68 dB at 4 weeks,
55 dB at 6 weeks, 50 dB at 13 weeks, and 38 dB at
9 × 10¹¹ vg (n=1) 1·5 × 10¹² vg (n=5)
Grade 1 Grade 2 Grade 3 Grade 1 Grade 2 Grade 3
Increased lymphocyte count 0 1 0 0 5 0
Decreased neutrophil count 0 0 0 0 3 2
Decreased haemoglobin 0 0 0 3 0 0
Increased lactate dehydrogenase 1 0 0 5 0 0
Hyperglycaemia 2 0 0 0 0 0
Increased triglycerides 1 0 0 0 0 0
Decreased haptoglobin 0 0 0 3 0 0
Increased cholesterol 0 0 0 1 0 0
Prolonged activated partial
thromboplastin time
0 0 0 3 0 0
Decreased fibrinogen 0 0 0 4 0 0
Influenza-like symptoms 1 0 0 0 0 0
COVID-19 0 0 0 2 0 0
Fever 0 0 0 7 0 0
Rhinobyon 0 0 0 1 0 0
Nausea 0 0 0 1 0 0
Decreased appetite 0 0 0 1 0 0
Constipation 0 0 0 1 0 0
No grade 4 or grade 5 adverse events occurred during the trial. vg=vector genomes.
Table 2: Summary of adverse events
Articles
www.thelancet.com Published online January 24, 2024 https://doi.org/10.1016/S0140-6736(23)02874-X 5
Figure: Audiometric test before and after inner ear administration of AAV1-hOTOF
Participant 1 received 9 × 10¹¹ vg AAV1-hOTOF; all others received 1·5 × 10¹² vg AAV1-hOTOF. Arrows indicate no response even at the maximum sound intensity level. Participants 4, 5, and 6 could not
complete pure-tone audiometry due to their young age. ABR=auditory brainstem response. ASSR=auditory steady-state response. PTA=pure-tone audiometry. vg=vector genomes.
0·2
5 0·5 1 2 4
140
120
100
80
60
40
20
0
Th
re
sh
ol
ds
(d
B)
Frequency (kHz)
ABR
0·2
5 0·5 1 2 4
Frequency (kHz)
0·2
5 0·5 1 2 4
Frequency (kHz)
0·2
5
0·1
25 0·2
5 0·5 1 2 4 80·5 1 2 4
Frequency (kHz) Frequency (kHz)
ASSR
E Participant 5
140
120
100
80
60
40
20
0
Th
re
sh
ol
ds
(d
B)
ABR ASSR PTA
C Participant 3
0·2
5 0·5 1 2 4
140
120
100
80
60
40
20
0
Th
re
sh
ol
ds
(d
B)
Frequency (kHz)
ABR
0·2
5 0·5 1 2 4
Frequency (kHz)
0·2
5 0·5 1 2 4
Frequency (kHz)
0·2
5 0·5 1 2 4
Frequency (kHz)
ASSR
F Participant 6
140
120
100
80
60
40
20
0
Th
re
sh
ol
ds
(d
B)
ABR ASSR
D Participant 4
0·2
5
Cli
ck 0·5 1 2 4
Frequency (kHz)
0·2
5
0·1
25 0·2
5 0·5 1 2 4 80·5 1 2 4
Frequency (kHz) Frequency (kHz)
0·2
5 0·5 1 2 4
140
120
100
80
60
40
20
0
Th
re
sh
ol
ds
(d
B)
ABR ASSR PTA
A Participant 1
Frequency (kHz)
0·2
5
0·1
25 0·2
5 0·5 1 2 4 80·5 1 2 4
Frequency (kHz) Frequency (kHz)
140
120
100
80
60
40
20
0
Th
re
sh
ol
ds
(d
B)
ABR ASSR PTA
B Participant 2
Baseline 4 weeks 6 weeks 13 weeks 26 weeks
Left ear Left ear
Participant 1 Participant 2 Participant 3 Participant 4 Participant 5 Participant 6
AAV1-neutralising antibodies
Baseline <1:5 1:35 <1:5 <1:5 <1:5 <1:5
6 weeks 1:405 1:3645 1:405 1:135 1:1215 1:405
13 weeks 1:1215 1:3645 1:1215 1:135 1:1215 1:1215
Interferon gamma
Baseline Negative Negative Negative Negative Negative Negative
6 weeks Negative Negative Negative Negative Negative Negative
13 weeks Negative Negative Negative Negative Negative Negative
Vector DNA
Baseline Negative Negative Negative Negative Negative Negative
1 week Negative Negative Negative Negative Negative Negative
Negative indicates that the T cell responses to the AAV1 capsid or vector DNA were below the lower limit of detection. AAV1=adeno-associated virus serotype 1.
Table 3: Immunity response and vector shedding
Articles
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26 weeks (figure D). The best recovery of ABR threshold
for participant 4 was 25 dB at 4 kHz at 26 weeks
(appendix p 16). The average auditory steady-state
response was gradually reduced to 50 dB at 26 weeks
from 100 dB at baseline. In participant 5, the average
ABR threshold was greater than 95 dB at baseline,
greater than 95 dB at 4 weeks, 65 dB at 6 weeks, 53 dB at
13 weeks, and 40 dB at 26 weeks (figure E). The best
recovery of ABR threshold for participant 5 was 25 dB at
0·25 kHz at 26 weeks (appendix p 16). The average
auditory steady-state response was gradually reduced to
40 dB at 26 weeks from greater than 98 dB at baseline. In
participant 6, the average ABR threshold was greater
than 95 dB at baseline, greater than 93 dB at 4 weeks,
greater than 90 dB at 6 weeks, 60 dB at 13 weeks, and
55 dB at 26 weeks (figure F). The average auditory
steady-state response was gradually reduced to 60 dB at
26 weeks from 100 dB at baseline. Pure-tone audiometry
was not done for participants 4, 5, and 6 because of their
young age.
The signal-to-noise ratio of the distortion product
otoacoustic emission test in participants 2, 4, 5, and 6
after treatment was lower at 4 weeks than at baseline
(appendix p 14). The signal-to-noise ratio showed recovery
in participants 2, 4, 5, and 6 in most frequencies. In
participant 3, we found no apparent change in the
signal-to-noise ratio after the treatment compared with
the baseline (appendix p 14).
Changes in scores of auditory and speech perception
from baseline to weeks 4, 13, and 26 are shown in table 4.
In participants 1, 3, and 5, with the cochlear implant
switched off, the Meaningful Auditory Integration Scale
score, the Categories of Auditory Performance score,
and the Meaningful Use of Speech Scale score were
MAIS or
IT-MAIS score
CAP score SIR score MUSS score Ambient sound
perception, %
Tone perception, % Initial
perception, %
Final
perception, %
Participant 1
Baseline 5 0 5 3 ND ND ND ND
4 weeks ND ND ND ND 56·3% 62·5% 37·5% 20·8%
13 weeks 15 4 5 6 75·0% 37·5% 20·8% 29·2%
26 weeks 30 7 5 37 100% 100% 83·3% 91·7%
Participant 2
Baseline 4 0 4 3 0 0 0 0
4 weeks 4 0 4 3 0 0 0 0
13 weeks 4 0 4 3 0 0 0 0
26 weeks 4 0 4 3 0 0 0 0
Participant 3
Baseline 6 1 5 3 0 0 0 0
4 weeks 13 4 5 5 46·9% 18·8% 18·8% 39·6%
13 weeks 13 4 5 5 50·0% 43·8% 12·5% 41·7%
26 weeks 20 7 5 40 93·8% 93·8% 54·2% 100%
Participant 4
Baseline 0 0 1 0 NA NA NA NA
4 weeks 2 2 1 0 NA NA NA NA
13 weeks 2 2 1 0 NA NA NA NA
26 weeks 17 2 2 3 NA NA NA NA
Participant 5
Baseline 2 0 3 2 0 0 0 0
4 weeks 4 2 3 2 18·8% 0 0 0
13 weeks 6 4 3 2 68·8% 0 0 8·3
26 weeks 23 6 4 39 87·5% 68·8% 62·5% 70·8%
Participant 6
Baseline 3 1 1 0 NA NA NA NA
4 weeks 13 3 1 5 NA NA NA NA
13 weeks 36 5 2 25 NA NA NA NA
26 weeks 36 6 2 30 NA NA NA NA
Participants 1, 2, 3, and 5 were tested with the cochlear implant switched off; participants 4 and 6 had no cochlear implants. MAIS was assessed in participants 1, 2, 3,
and 5. IT-MAIS was assessed in participants 4 and 6. Participants 4 and 6 were too young to complete tests for speech perception. Perception of ambient sound, tone, initial,
and final were assessed in a quiet environment. CAP=Categories of Auditory Performance. IT-MAIS=Infant-Toddler Meaningful Auditory Integration Scale. MAIS=Meaningful
Auditory Integration Scale. MUSS=Meaningful Use of Speech Scale. NA=not applicable. ND=not done. SIR=Speech Intelligibility Rating.
Table 4: Scores of auditory and speech perception (without cochlear implant or with the cochlear implant switched off)
Articles
www.thelancet.com Published online January 24, 2024 https://doi.org/10.1016/S0140-6736(23)02874-X 7
improved by 26 weeks; in a quiet environment, the
perception of ambient sound, tone, initial, and final was
also improved by 26 weeks (table 4). In participants 4
and 6, without the cochlear implant, the Infant–Toddler
Meaningful Auditory Integration Scale score, the
Categories of Auditory Performance score, and the
Meaningful Use of Speech Scale score, and the Speech
Intelligibility Rating score, were improved by 26 weeks
(table 4). Participant 1, with the cochlear implant
switched off, was unable to recognise speech in steady-
state noise or to complete the assessment for speech
recognition thresholds of monosyllable, disyllable, and
sentence conditions at baseline, 4 weeks, and 13 weeks
(appendix p 19). However, at 26 weeks, her speech
recognition thresholds in steady-state noise were
improved in the monosyllable (–2·0 dB), disyllable
(0·3 dB), and sentence (8·9 dB) conditions (appendix
p 19). No improvements in auditory or speech perception
were observed in participant 2 (table 4). Participant 3’s
perception of monosyllabic words was 0% at baseline
and 74·0% at 26 weeks, perception of disyllabic words
was 0% at baseline and 88·6% at 26 weeks, and
perception of sentences was 0% at baseline and 73·6% at
26 weeks (appendix p 19). Participant 3, with the cochlear
implant switched off, was unable to recognise speech in
steady-state noise and unable to complete the assessment
for speech recog nition thresholds in the monosyllable,
disyllable, and sentence conditions at baseline, 4 weeks,
and 13 weeks (appendix p 19). However, at 26 weeks, her
speech recog nition thresholds in steady-state noise were
improved in the monosyllable (6·4 dB), disyllable
(9·7 dB), and sentence (29·0 dB) conditions (appendix
p 19). Repre sentative speech communication of
participant 3 after treatment is presented in video 1.
Representative speech communication of participant 4
after treatment is presented in video 2.
Participant 5, with the cochlear implant switched off,
she was unable to recognise speech in quiet environment
at baseline, 4 weeks, and 13 weeks (appendix p 19).
However, at 26 weeks, her speech perception in a quiet
environment was improved in the monosyllable (54·0%),
disyllable (62·9%), and sentence (23·6%) conditions
(appendix p 19). Representative speech communication
of participant 5 after treatment is presented in video 3.
Representative speech communication of participant 6
after treatment is presented in video 4.
Results for auditory and speech perception with the
cochlear implant switched on (for participants 1, 3, and 5;
participant 2 had no improvements in response) are
shown in appendix (pp 18–20).
Discussion
In this trial, no dose-limiting toxicity was recorded during
26 weeks of follow-up after unilateral injection of
AAV1-hOTOF at 9 × 10¹¹ vg or 1·5 × 10¹² vg. AAV1 has been
previously used as a vector for gene therapy for lipoprotein
lipase deficiency.26 To further improve the safety profile of
AAV1 for this study, a hair-cell specific promoter was used
to minimise ectopic expression of otoferlin. Participants
were given dexamethasone to minimise the risk of
inflammation and ceftriaxone to minimise the risk of
infection. Neither aural inflammation nor T cell responses
to the AAV1 capsid were observed. 46 (96%) of all
48 adverse events were grade 1–2 and two (4%) were
grade 3. None of the observed adverse events met Hy’s
law criteria for liver injury, an important concern with
gene therapy.27 We found no evidence that adverse events
affected the treatment outcome. Although two
participants had COVID-19 (participant 4 at 2 weeks after
injection and participant 5 at 1 week after injection), their
hearing was recovered by 4–6 weeks after injection.
Altogether, these findings suggest that, with concomitant
anti-inflam matory treatment, local and systemic
inflammatory responses can be reduced to an acceptable
level.
Our efficacy assessment revealed robust hearing
recovery in all but one participant. Hearing recovery was
first detected 4–6 weeks after injection in participants 1, 3,
4, 5, and 6. A key finding in this trial is a time-dependent
hearing recovery, and participants are being followed up
to further verify the temporal pattern. Treatment efficacy
did not depend on vector dosage administered, but the
number of participants included in this analysis is too
small for any meaningful interpretation of a dose–
response relationship, and further investigation in larger
randomised trials would be required to generate adequate
evidence regarding a dose–response relationship of treat-
ment with AAV1-hOTOF gene therapy.
The signal-to-noise ratio of the distortion product
otoacoustic emission test showed reductions at 4 weeks
after treatment in five participants (participants 1, 2, 4, 5,
and 6) (appendix p 14). The fairly stable signal-to-noise
ratio of the distortion product otoacoustic emission test
before and after injection in participant 3 might be due to
less damage to the round window during surgery or to a
reduced inflammatory reaction to AAV1-hOTOF com-
pared with other participants. The signal-to-noise ratio of
the distortion product otoacoustic emission test in
participant 3 might also have changed before the first
follow-up but recovered quickly after the injection, which
could mean that change could not be detected at 4 weeks.
Overall, the signal-to-noise ratio of the distortion product
otoacoustic emission test of most participants decreased,
followed by a recovery, although the degree of recovery
varied among participants.
Children with congenital deafness face difficulties
when learning spoken language because of not being
able to hear spoken sounds; after hearing recovery, such
children gradually acquire the ability of speech
perception.28,29 Therefore, besides objective audiometric
tests, speech perception is also an important indicator of
hearing recovery in children with hearing loss. Testing
for speech perception showed improvement in all
responding participants (participants 1, 3, 4, 5, and 6).
See Online for video 1–4
Articles
8 www.thelancet.com Published online January 24, 2024 https://doi.org/10.1016/S0140-6736(23)02874-X
Participants 1, 3, and 5 had improvements in auditory
and speech perception with the cochlear implant off by
26 weeks. At 4 and 13 weeks, with the cochlear implant
off, participants 1 and 3 were unable to recognise speech
in a noisy environment; however, by 26 weeks, both
participants were able to recognise speech in a noisy
environment and communicate using the telephone
without difficulties. Participant 5, with the cochlear
implant switched off, was also able to have a spoken
conversation without difficulties (video 3). Children with
cochlear implants generally need 1·0–1·5 years of speech
rehabilitation to achieve good improvement in sound
perception and speech recognition.30–32 The improvement
in participants 1, 3, and 5 might be partly due to the
continuous hearing recovery after gene therapy and the
benefit of speech rehabilitation. Participants 4 and 6 did
not receive a cochlear implant and scored 0 on all
measures of speech perception before the injection. After
injection, speech perception was improved to different
degrees in participants 4 and 6, which might be caused
by differences in the participants’ individual abilities or
different speech rehabilitation education. Notably,
children with autosomal recessive deafness 9 might need
time to further develop speech perception after an
improvement in hearing, and the development of speech
or language skills is variable from child to child. Some
participants were too young to complete some tests. In
the future, more objective and comprehensive speech
assessments need to be developed and explored.
Inner-ear injection through the round window
membrane is a common surgical approach to deliver
AAV vectors in mice and non-human primates.33,34
However, to our knowledge, no study has investigated
the same approach to deliver AAV vectors to the human
cochlea. In this trial, AAV1-hOTOF was injected through
the round window membrane via the external auditory
canal under direct vision with an endoscope rather than
cortical mastoidectomy to minimise the risk of damage
to the mastoid cavity and tympanic sinus. Injection via
the round window membrane without fenestration
might lead to variable hair-cell transduction efficiency
along the tonotopic positions inside the cochlea and can
carry a risk of injection-induced hearing loss.35,36 To
further minimise the risk and improve the transduction
efficiency, we used a round window membrane injection
with a small fenestration introduced to the stapes
footplate to promote lymph flow.
The auditory function of participant 2 was not improved
by 26 weeks after injection, for reasons that could not be
established. One possibility might be the higher
concentrations of neutralising antibodies at baseline
(1:135 in participant 2, compared with <1:5 in other
participants) and after treatment (1:3645 at 6 weeks), as
previously reported for AAV-mediated gene therapy.37–39 An
alternative explanation is a possible leakage of the
AAV1-hOTOF solution from the round window membrane
during or after surgery.
In conclusion, we found that a single injection of
AAV1-hOTOF resulted in robust hearing recovery in five
of six children with autosomal recessive deafness 9, and
in improved speech perception in those who had hearing
recovery, without dose-limiting toxicities at either
administered dose. This study supports the continuous
investigation of gene therapy to treat hearing loss in
children with autosomal recessive deafness 9. Trials with
larger sample sizes and longer follow-ups are needed to
further examine the efficacy of gene therapy compared
with that of cochlear implants.
Contributors
YS and HL were the principal investigators of the study and conceived
the trial. YS, ZC, JL, HW, XC, YC, DW, HT, and KG contributed to the
study design. JL, HW, and QC enrolled participants. JL, HW, XC, QC,
and YY collected the data. XC collected the questionnaires. QC prepared
the videos. JL, HW, XC, YC, DW, YS, ZC, and KG analyed and interpreted
data and wrote the manuscript. JL, HW, XC, YC, DW,
YS, and RC accessed and verified the data. SH, BZ, RC, HT, CC, LJ, and
ZG contributed to the revision of the manuscript. YS, HL, ZC, and YC
obtained funding. YS, WW, HL, BC, JL, HW, and LZ participated in the
surgery. MX, ZW, JW, and JZ processed the blood sample. LG, SY, DW,
and HT confirmed the genotype of participants. All authors vouch for the
fidelity of the protocol and the accuracy and completeness of the reported
data. All authors reviewed and approved of the manuscript before
submission. JL, HW, XC, YC, DW, YS, and ZC had full access to all data
in the study and had final responsibility for the decision to submit for
publication.
Declaration of interests
KG is a staff of the Shanghai Refreshgene Therapeutics. ZC is a
cofounder of Salubritas Therapeutics. All other authors declare no
competing interests.
Data sharing
To respect the privacy of participants, individual participant data is
anonymised. De-identified data (text, figures, tables, and appendices) in
the manuscript are available. The redacted trial protocol is available in
the appendix. These data will be available from the corresponding
author.
Acknowledgments
The Eye & ENT Hospital of Fudan University sponsored the study. The
study was supported by the National Natural Science Foundation of
China (82225014, 82171148, and 82192864), the National Key R&D
Program of China (2020YFA0908201, 2021YFA1101302, and
2023YFC2508400), Science and Technology Commission of Shanghai
Municipality (21S11905100), Shanghai Municipal Health Commission
(20224Z0003), Shanghai Municipal Education Commission
(2023ZKZD12), and Fudan University (yg2022-23). The study was also
funded by Shanghai Refreshgene Therapeutics. ZC was supported by
the Ines and Fredrick Yeatts Fund. We thank the participants and their
families for support of the study. We thank the physicians and staff at
the Eye & ENT Hospital of Fudan University for laboratory testing,
audiometric examination, aural endoscopy, and vestibular function
examination, and the nurses for professional care of participants during
hospitalisation. We thank Yongfu Yu from Fudan University for assisting
with project design. Writing and editorial assistance was provided by
Kehong Zhang from Ivy Medical Editing (Shanghai, China).
References
1 Morton CC, Nance WE. Newborn hearing screening—a silent
revolution. N Engl J Med 2006; 354: 2151–64.
2 Spencer L. Global, regional, and national incidence, prevalence,
and years lived with disability for 354 diseases and injuries for
195 countries and territories, 1990-2017: a systematic analysis for
the Global Burden of Disease Study 2017. Lancet 2018;
392: 1789–858.
3 Roux I, Safieddine S, Nouvian R, et al. Otoferlin, defective in a
human deafness form, is essential for exocytosis at the auditory
ribbon synapse. Cell 2006; 127: 277–89.
Articles
www.thelancet.com Published online January 24, 2024 https://doi.org/10.1016/S0140-6736(23)02874-X 9
4 Sloan-Heggen CM, Bierer AO, Shearer AE, et al. Comprehensive
genetic testing in the clinical evaluation of 1119 patients with
hearing loss. Hum Genet 2016; 135: 441–50.
5 Rodríguez-Ballesteros M, Reynoso R, Olarte M, et al. A multicenter
study on the prevalence and spectrum of mutations in the otoferlin
gene (OTOF) in subjects with nonsyndromic hearing impairment
and auditory neuropathy. Hum Mutat 2008; 29: 823–31.
6 Iwasa YI, Nishio SY, Sugaya A, et al. OTOF mutation analysis with
massively parallel DNA sequencing in 2265 Japanese sensorineural
hearing loss patients. PLoS One 2019; 14: e0215932.
7 Choi BY, Ahmed ZM, Riazuddin S, et al. Identities and frequencies
of mutations of the otoferlin gene (OTOF) causing DFNB9 deafness
in Pakistan. Clin Genet 2009; 75: 237–43.
8 Gallo-Terán J, Megía López R, Morales-Angulo C, et al. Estudio de
una familia con hipoacusia neurosensorial secundaria a la
mutación q829x en el gen otof. Acta Otorrinolaringol Esp 2004;
55: 120–25.
9 Migliosi V, Modamio-Høybjør S, Moreno-Pelayo MA, et al. Q829X,
a novel mutation in the gene encoding otoferlin (OTOF),
is frequently found in Spanish patients with prelingual non-
syndromic hearing loss. J Med Genet 2002; 39: 502–06.
10 Bainbridge JW, Mehat MS, Sundaram V, et al. Long-term effect of
gene therapy on Leber’s congenital amaurosis. N Engl J Med 2015;
372: 1887–97.
11 Chowdary P, Shapiro S, Makris M, et al. Phase 1-2 trial of AAVS3
gene therapy in patients with hemophilia B. N Engl J Med 2022;
387: 237–47.
12 Jiang L, Wang D, He Y, Shu Y. Advances in gene therapy hold
promise for treating hereditary hearing loss. Mol Ther 2023;
31: 934–50.
13 Akil O, Dyka F, Calvet C, et al. Dual AAV-mediated gene therapy
restores hearing in a DFNB9 mouse model. Proc Natl Acad Sci USA
2019; 116: 4496–501.
14 Al-Moyed H, Cepeda AP, Jung S, Moser T, Kügler S, Reisinger E.
A dual-AAV approach restores fast exocytosis and partially rescues
auditory function in deaf otoferlin knock-out mice. EMBO Mol Med
2019; 11: e9396.
15 Tang H, Wang H, Wang S, et al. Hearing of Otof-deficient mice
restored by trans-splicing of N- and C-terminal otoferlin.
Hum Genet 2023; 142: 289–304.
16 Zhang L, Wang H, Xun M, et al. Preclinical evaluation of the
efficacy and safety of AAV1-hOTOF in mice and nonhuman
primates. Mol Ther Methods Clin Dev 2023; 31: 101154.
17 WHO. World report on hearing. Geneva: World Health
Organization, 2021. https://www.who.int/publications/i/
item/9789240020481 (accessed Dec 1, 2022).
18 Robbins AM, Renshaw JJ, Berry SW. Evaluating meaningful
auditory integration in profoundly hearing-impaired children.
Am J Otol 1991; 12 (suppl): 144–50.
19 Archbold S, Lutman ME, Nikolopoulos T. Categories of auditory
performance: inter-user reliability. Br J Audiol 1998; 32: 7–12.
20 Cox RM, McDaniel DM. Development of the Speech Intelligibility
Rating (SIR) test for hearing aid comparisons. J Speech Hear Res
1989; 32: 347–52.
21 Robbins AM, Osberger MJ. Meaningful Use of Speech Scale
(MUSS). Indianopolis: Indiana University School of Medicine,
1990.
22 Fu Q J, Zhu M, Wang X. Development and validation of the
Mandarin speech perception test. J Acoust Soc Am 2011;
129: EL267–73.
23 Cheng X, Liu Y, Shu Y, et al. Music training can improve music and
speech perception in pediatric mandarin-speaking cochlear implant
users. Trends Hear 2018; 22: 2331216518759214.
24 Tao D, Deng R, Jiang Y, Galvin JJ 3rd, Fu Q J, Chen B. Melodic pitch
perception and lexical tone perception in Mandarin-speaking
cochlear implant users. Ear Hear 2015; 36: 102–10.
25 Chandrasekhar SS, Tsai Do BS, Schwartz SR, et al. Clinical practice
guideline: sudden hearing loss (update). Otolaryngol Head Neck Surg
2019; 161: S1–45.
26 Mingozzi F, Meulenberg JJ, Hui DJ, et al. AAV-1-mediated gene
transfer to skeletal muscle in humans results in dose-dependent
activation of capsid-specific T cells. Blood 2009; 114: 2077–86.
27 US Food and Drug Administration. Guidance for industry.
Drug-induced liver injury: premarking clinical evaluation. Rockville,
MD: Food and Drug Administration, 2009. https://www.fda.gov/
media/116737/download (accessed June 1, 2023).
28 Dornhoffer JR, Reddy P, Meyer TA, Schvartz-Leyzac KC, Dubno JR,
McRackan TR. Individual differences in speech recognition changes
after cochlear implantation. JAMA Otolaryngol Head Neck Surg 2021;
147: 280–86.
29 Benchetrit L, Ronner EA, Anne S, Cohen MS. Cochlear
implantation in children with single-sided deafness: a systematic
review and meta-analysis. JAMA Otolaryngol Head Neck Surg 2021;
147: 58–69.
30 Santarelli R, Scimemi P, Costantini M, Domínguez-Ruiz M,
Rodríguez-Ballesteros M, Del Castillo I. Cochlear synaptopathy due
to mutations in OTOF gene may result in stable mild hearing loss
and severe impairment of speech perception. Ear Hear 2021;
42: 1627–39.
31 Santarelli R, del Castillo I, Cama E, Scimemi P, Starr A. Audibility,
speech perception and processing of temporal cues in ribbon
synaptic disorders due to OTOF mutations. Hear Res 2015;
330: 200–12.
32 Zheng D, Liu X. Cochlear implantation outcomes in patients with
OTOF mutations. Front Neurosci 2020; 14: 447.
33 Zhao Y, Zhang L, Wang D, Chen B, Shu Y. Approaches and vectors
for efficient cochlear gene transfer in adult mouse models.
Biomolecules 2022; 13: 38.
34 Akil O, Seal RP, Burke K, et al. Restoration of hearing in the
VGLUT3 knockout mouse using virally mediated gene therapy.
Neuron 2012; 75: 283–93.
35 Chien WW, McDougald DS, Roy S, Fitzgerald TS, Cunningham LL.
Cochlear gene transfer mediated by adeno-associated virus:
comparison of two surgical approaches. Laryngoscope 2015;
125: 2557–64.
36 Yoshimura H, Shibata SB, Ranum PT, Smith RJH. Enhanced viral-
mediated cochlear gene delivery in adult mice by combining canal
fenestration with round window membrane inoculation. Sci Rep
2018; 8: 2980.
37 Jiang H, Couto LB, Patarroyo-White S, et al. Effects of transient
immunosuppression on adenoassociated, virus-mediated, liver-
directed gene transfer in rhesus macaques and implications for
human gene therapy. Blood 2006; 108: 3321–28.
38 Manno CS, Pierce GF, Arruda VR, et al. Successful transduction of
liver in hemophilia by AAV-Factor IX and limitations imposed by
the host immune response. Nat Med 2006; 12: 342–47.
39 Wang L, Herzog RW. AAV-mediated gene transfer for treatment of
hemophilia. Curr Gene Ther 2005; 5: 349–60.