FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2570201053> ?p ?o ?g. }

• W2570201053 endingPage "391" @default.
• W2570201053 startingPage "379" @default.
• W2570201053 abstract "Adeno-associated virus (AAV) is a safe and effective vector for gene therapy for retinal disorders. Gene therapy for hearing disorders is not as advanced, in part because gene delivery to sensory hair cells of the inner ear is inefficient. Although AAV transduces the inner hair cells of the mouse cochlea, outer hair cells remain refractory to transduction. Here, we demonstrate that a vector, exosome-associated AAV (exo-AAV), is a potent carrier of transgenes to all inner ear hair cells. Exo-AAV1-GFP is more efficient than conventional AAV1-GFP, both in mouse cochlear explants in vitro and with direct cochlear injection in vivo. Exo-AAV shows no toxicity in vivo, as assayed by tests of auditory and vestibular function. Finally, exo-AAV1 gene therapy partially rescues hearing in a mouse model of hereditary deafness (lipoma HMGIC fusion partner-like 5/tetraspan membrane protein of hair cell stereocilia [Lhfpl5/Tmhs−/−]). Exo-AAV is a powerful gene delivery system for hair cell research and may be useful for gene therapy for deafness. Adeno-associated virus (AAV) is a safe and effective vector for gene therapy for retinal disorders. Gene therapy for hearing disorders is not as advanced, in part because gene delivery to sensory hair cells of the inner ear is inefficient. Although AAV transduces the inner hair cells of the mouse cochlea, outer hair cells remain refractory to transduction. Here, we demonstrate that a vector, exosome-associated AAV (exo-AAV), is a potent carrier of transgenes to all inner ear hair cells. Exo-AAV1-GFP is more efficient than conventional AAV1-GFP, both in mouse cochlear explants in vitro and with direct cochlear injection in vivo. Exo-AAV shows no toxicity in vivo, as assayed by tests of auditory and vestibular function. Finally, exo-AAV1 gene therapy partially rescues hearing in a mouse model of hereditary deafness (lipoma HMGIC fusion partner-like 5/tetraspan membrane protein of hair cell stereocilia [Lhfpl5/Tmhs−/−]). Exo-AAV is a powerful gene delivery system for hair cell research and may be useful for gene therapy for deafness. Hearing loss, congenital or acquired (most commonly age-related hearing loss), is a major health issue that affects approximately 30 million people in the United States alone1Lin F.R. Niparko J.K. Ferrucci L. Hearing loss prevalence in the United States.Arch. Intern. Med. 2011; 171: 1851-1852Crossref PubMed Scopus (491) Google Scholar compared to about 5.4 million for Alzheimer’s disease.2Alzheimer’s Association2016 Alzheimer’s disease facts and figures.Alzheimers Dement. 2016; 12: 459-509Abstract Full Text Full Text PDF PubMed Scopus (1795) Google Scholar Congenital hearing loss has an incidence of about 1:1,000 births,3Mason J.A. Herrmann K.R. Universal infant hearing screening by automated auditory brainstem response measurement.Pediatrics. 1998; 101: 221-228Crossref PubMed Scopus (239) Google Scholar of which about half have a defined genetic cause. Because the cochlea is surgically accessible and local application into a relatively immune-protected environment is possible, gene therapy using viral vectors is an attractive approach for treating hearing loss. For congenital recessive deafness, gene addition is possible, whereas congenital dominant forms might be treated by silencing or correcting the mutated gene.4Géléoc G.S. Holt J.R. Sound strategies for hearing restoration.Science. 2014; 344: 1241062Crossref PubMed Scopus (145) Google Scholar Gene therapy also holds promise for age-related hearing loss by targeting pathways involved in hair cell (HC) or spiral ganglion neuron survival (e.g., neurotrophic factors5Wise A.K. Tu T. Atkinson P.J. Flynn B.O. Sgro B.E. Hume C. O’Leary S.J. Shepherd R.K. Richardson R.T. The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection.Hear. Res. 2011; 278: 69-76Crossref PubMed Scopus (47) Google Scholar or antioxidant proteins6Kawamoto K. Sha S.H. Minoda R. Izumikawa M. Kuriyama H. Schacht J. Raphael Y. Antioxidant gene therapy can protect hearing and hair cells from ototoxicity.Mol. Ther. 2004; 9: 173-181Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 7Kawamoto K. Yagi M. Stöver T. Kanzaki S. Raphael Y. Hearing and hair cells are protected by adenoviral gene therapy with TGF-beta1 and GDNF.Mol. Ther. 2003; 7: 484-492Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) or by manipulating gene expression in supporting cells to induce their transdifferentiation into hair cells.8Li W. Wu J. Yang J. Sun S. Chai R. Chen Z.Y. Li H. Notch inhibition induces mitotically generated hair cells in mammalian cochleae via activating the Wnt pathway.Proc. Natl. Acad. Sci. USA. 2015; 112: 166-171Crossref PubMed Scopus (120) Google Scholar For congenital hereditary hearing loss, at least 70 genes are causative in humans. In many cases, they affect the function of hair cells, the receptor cells of the inner ear. The cochlea has two types of hair cells. Inner hair cells (IHCs) convert the mechanical stimulus of sound vibration into a neural signal that is transmitted by type I spiral ganglion neurons to the brain. Outer hair cells (OHCs) connect only to poorly defined type II neurons. Their main function is to amplify the vibration produced by sound by as much as 60 decibels (dB) in a frequency-specific manner, and they are essential for frequency discrimination9Liberman M.C. Gao J. He D.Z. Wu X. Jia S. Zuo J. Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier.Nature. 2002; 419: 300-304Crossref PubMed Scopus (683) Google Scholar (important in speech perception). Most deafness genes known to affect hair cell function are expressed in both cell types, so, in general, gene therapy strategy should target both IHCs and OHCs. Hair cells in the vestibular system are essential for our sense of balance and for coordinating eye movements. They are often affected in hereditary deafness so gene therapies should target them as well. The major limitation of gene therapy for the cochlea is the relative inefficiency of vectors that mediate transgene expression in hair cells. Several gene delivery strategies have targeted sensory cells in the cochlea, including viral and non-viral methods (see Kohrman and Raphael10Kohrman D.C. Raphael Y. Gene therapy for deafness.Gene Ther. 2013; 20: 1119-1123Crossref PubMed Scopus (12) Google Scholar and Sacheli et al.11Sacheli R. Delacroix L. Vandenackerveken P. Nguyen L. Malgrange B. Gene transfer in inner ear cells: a challenging race.Gene Ther. 2013; 20: 237-247Crossref PubMed Scopus (45) Google Scholar for review). As yet, however, none of these has led to efficient transgene expression in hair cells. Adeno-associated virus (AAV) vectors are presently the most promising vectors for cochlear gene delivery, but in vivo transduction is mostly limited to IHCs. In previous studies,12Akil O. Seal R.P. Burke K. Wang C. Alemi A. During M. Edwards R.H. Lustig L.R. Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.Neuron. 2012; 75: 283-293Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 13Askew C. Rochat C. Pan B. Asai Y. Ahmed H. Child E. Schneider B.L. Aebischer P. Holt J.R. Tmc gene therapy restores auditory function in deaf mice.Sci. Transl. Med. 2015; 7: 295ra108Crossref PubMed Scopus (166) Google Scholar virtually no OHCs were transduced by AAV vectors after in vivo injection in mice. Similarly, it is difficult to express genes in hair cells in vitro for research, which has slowed characterization of proteins involved in hair cell function. There is, therefore, a great need for a vector system that effectively transduces both IHCs and OHCs, both in vitro and in vivo. It would pave the way to clinical trials, and would also be useful in studying hair cell physiology. In this work, we tested exosome-associated AAV vectors (exo-AAVs) for delivery to cochlear hair cells and compared different injection routes to the cochlea in mice. Exosomes are cell-derived natural lipid structures involved in intercellular communication and are potential therapeutic carriers of nucleic acids and proteins (see Fitzpatrick et al.14Fitzpatrick Z. György B. Skog J. Maguire C.A. Extracellular vesicles as enhancers of virus vector-mediated gene delivery.Hum. Gene Ther. 2014; 25: 785-786Crossref PubMed Scopus (12) Google Scholar and György et al.15György B. Hung M.E. Breakefield X.O. Leonard J.N. Therapeutic applications of extracellular vesicles: clinical promise and open questions.Annu. Rev. Pharmacol. Toxicol. 2015; 55: 439-464Crossref PubMed Scopus (356) Google Scholar). We previously observed that exosome association of AAV enhances transduction of cells in vitro and in vivo,16György B. Fitzpatrick Z. Crommentuijn M.H. Mu D. Maguire C.A. Naturally enveloped AAV vectors for shielding neutralizing antibodies and robust gene delivery in vivo.Biomaterials. 2014; 35: 7598-7609Crossref PubMed Scopus (91) Google Scholar, 17Maguire C.A. Balaj L. Sivaraman S. Crommentuijn M.H. Ericsson M. Mincheva-Nilsson L. Baranov V. Gianni D. Tannous B.A. Sena-Esteves M. et al.Microvesicle-associated AAV vector as a novel gene delivery system.Mol. Ther. 2012; 20: 960-971Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar so we hypothesized that exosomes would also augment gene delivery into cochlear hair cells. We demonstrate here that exo-AAV vectors are efficient carriers of transgenes into cochlear and vestibular hair cells both in vitro and in vivo. Exo-AAV vectors outperform conventional AAV vectors in gene transfer efficiency and are well tolerated. Furthermore, we show that exo-AAV is useful as a gene therapy system, partially rescuing hearing in a mouse model of human deafness (lipoma HMGIC fusion partner-like 5/tetraspan membrane protein of hair cell stereocilia [Lhfpl5/Tmhs−/−]). Of AAV serotypes, AAV1 (the number denotes the capsid serotype) has been reported to be the most effective for cochlear hair cell transduction in preclinical gene therapy studies.12Akil O. Seal R.P. Burke K. Wang C. Alemi A. During M. Edwards R.H. Lustig L.R. Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.Neuron. 2012; 75: 283-293Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 13Askew C. Rochat C. Pan B. Asai Y. Ahmed H. Child E. Schneider B.L. Aebischer P. Holt J.R. Tmc gene therapy restores auditory function in deaf mice.Sci. Transl. Med. 2015; 7: 295ra108Crossref PubMed Scopus (166) Google Scholar We therefore prepared exo-AAV1 and conventional AAV1. AAV1 and exo-AAV1 were isolated from the lysate and culture medium of vector-producing 293T cells, respectively (Figure 1A; see Materials and Methods). Using cryoelectron microscopy (cryo-EM) and transmission EM (TEM) in combination with immunogold labeling, we qualitatively observed AAV capsids both bound to the surface of exosomes and in their interiors (Figures 1A and S1A–S1D). The capsids lining the outer exosome membrane could be clearly distinguished as surface bound, whereas capsids that appear “inside” could be above, inside, or below the vesicle within the TEM section. However, we observed instances of capsids distorting the membrane from the interior of the vesicle, directly confirming that at least some of the capsids are indeed on the interior (Figure S1B). We first assessed transgene delivery efficiency of conventional AAV1 and exo-AAV1 vectors on cochlear explant cultures. Cochleas were dissected at postnatal day 1 (P1) and placed in organ culture. Vectors were added to the culture medium 1 day later. We used vectors encoding GFP under the strong hybrid cytomegalovirus (CMV)/chicken beta actin (CBA) promoter. After culturing the cochleas with the vectors for 3 days (equivalent to age P5), tissues were fixed, labeled with phalloidin and with antibodies to the hair cell marker myosin VIIa, and viewed with a confocal microscope. Exo-AAV1 vector was superior to conventional AAV1 vector in gene delivery to hair cells (Figures 1B–1E). At 1011 genomic copies (GCs) per cochlea, conventional AAV1-GFP vector transduced approximately 20% of IHCs and OHCs, whereas exo-AAV1-GFP transduced up to 65% of IHCs and 50% of OHCs (p < 0.001 and p < 0.01, respectively; Figure 1C). We also looked for regional differences in efficiency, and found exo-AAV1 outperformed conventional AAV1 at both the middle and basal turns of the cochlea (Figure 1D; p < 0.01 for middle and basal turns, not significant for apical turn). We also tested another serotype, AAV9, as both exo-AAV9 and conventional AAV9. At 1011 GCs/cochlea, we observed a significant enhancement of transduction by exo-AAV9 (Figures 1E and S2A). Strikingly, exo-AAV9 transduced almost 95% of IHCs and OHCs (Figure 1E). We conclude that both exo-AAV1 and exo-AAV9 vectors significantly enhance both IHC and OHC transduction in cochlear explant cultures compared to conventional AAV vectors. These results indicate that exo-AAVs can be powerful gene-delivery vehicles for in vitro experimental work, but they may not reflect the in vivo performance needed for therapy. We therefore compared conventional AAV1 and exo-AAV1 in vivo using direct injections into P0/P1 mouse cochleas. We compared two injection routes: through the round window membrane (RWM) into the scala tympani (used in two animal models of hereditary deafness12Akil O. Seal R.P. Burke K. Wang C. Alemi A. During M. Edwards R.H. Lustig L.R. Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.Neuron. 2012; 75: 283-293Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 13Askew C. Rochat C. Pan B. Asai Y. Ahmed H. Child E. Schneider B.L. Aebischer P. Holt J.R. Tmc gene therapy restores auditory function in deaf mice.Sci. Transl. Med. 2015; 7: 295ra108Crossref PubMed Scopus (166) Google Scholar), and through the lateral wall of the cochlea by cochleostomy at the basal turn. In both cases, 250 nL of virus-containing solution was injected over ∼10 min. Conventional AAV1-GFP transduced some IHCs but few OHCs. Cochleostomy delivery of conventional AAV1 vector resulted in only 36% GFP-positive IHCs and 17% GFP-positive OHCs (Figures 2A and 2B). Upon RWM injection, we observed more GFP-positive IHCs (up to 65%) but fewer OHCs (∼14%) (Figures 2A and 2B). With either route, however, exo-AAV1-GFP vectors significantly enhanced gene delivery. When delivered by cochleostomy, exo-AAV transduced 63% of IHCs and 28% of OHCs. With RWM injection, 88% of IHCs and 25% of OHCs were transduced (Figures 2A and 2B). As reported by Askew et al.,13Askew C. Rochat C. Pan B. Asai Y. Ahmed H. Child E. Schneider B.L. Aebischer P. Holt J.R. Tmc gene therapy restores auditory function in deaf mice.Sci. Transl. Med. 2015; 7: 295ra108Crossref PubMed Scopus (166) Google Scholar cochlear injection of P0 mouse pups is difficult, causing significant variability in transduction. Our results (Figures 2B–2D) include all of the injected mice (n = 38 for cochleostomy; n = 23 for RWM), even those with very low transduction efficiency that may result from unsuccessful injection. In our best injections with exo-AAV vectors, we observed >95% IHCs and ∼50% OHCs transduced with our working dose (250 nL, containing 5 × 109 genomic copies of AAV). The best cases of the cochleostomy and RWM injections had similar hair cell transduction rates. However, in our hands, cochleostomy results were more variable and there were more instances with very low GFP expression. Because GFP expression in individual hair cells may vary with multiple AAV genomes being delivered, we quantified GFP intensity using automated image analysis. Among GFP-positive IHCs, average GFP fluorescence intensity per cell was 70% higher with exo-AAV than with conventional AAV, with either cochleostomy or RWM injection (p < 0.01 for cochleostomy and p < 0.05 for RWM injection; Figure 2C). For OHCs, no significant difference in GFP intensity per cell was evident between exo-AAV1 and conventional AAV1. For cochleostomy, transduction rates varied with distance from the injection site. We counted more transduced hair cells in the base (near the injection site) than in the apex (Figure 2D). The gradient was particularly steep and significant for OHCs, with only a few OHCs transduced at the apex (repeated-measures ANOVA for the entire dataset to analyze the relationship between location and transduction; p = 0.0009 for AAV1 and p = 0.02 for exo-AAV1). With RWM injection, however, there was no significant gradient, suggesting that the virus can diffuse more freely with this approach. Overall, in all subregions tested, with both injection routes, exo-AAV1 significantly outperformed conventional AAV1 (Figure 2D). With cochleostomy injections of either conventional or exo-AAV1, we also observed robust expression of GFP in spiral ganglion neurons, cells in the inner sulcus, Claudius cells, and Hensen cells (Figure S3). Surprisingly, GFP-positive hair cells were also evident in the utricle and in the ampullas of the lateral semicircular canals following exo-AAV administration by either cochleostomy (Figure S4) or RWM injection (Figures 3A and 3B). In the utricle after RWM injection, exo-AAV1 transduced 30% of hair cells (Figure 3C), 2.3 times more than conventional AAV1 (p < 0.05, Mann Whitney U test), indicating some level of diffusion throughout endolymphatic compartments at that age. Several myosinVIIa-negative cells (supporting cells) were also transduced with either vector. Thus, exo-AAV vectors also have the potential for gene delivery to the vestibular system. Because exo-AAV9 outperformed exo-AAV1 in vitro, we also tested AAV9 in vivo. Transduction rates in vivo were similar between exo-AAV1 and exo-AAV9, with AAV9 targeting 60% of IHCs and 25% of OHCs after injection (Figure S2B). We next tested the ability of exo-AAV to produce efficient expression of a biologically relevant gene, asking whether exo-AAV1 could improve hearing in a mouse model for human hereditary deafness. We selected a mouse with a targeted deletion of Lhfpl5 (also known as Tmhs). LHFPL5 protein is an integral component of the mechanotransduction machinery in both OHCs and IHCs, and its absence leads to early hair cell degeneration, profound deafness, and severe vestibular dysfunction.18Xiong W. Grillet N. Elledge H.M. Wagner T.F. Zhao B. Johnson K.R. Kazmierczak P. Müller U. TMHS is an integral component of the mechanotransduction machinery of cochlear hair cells.Cell. 2012; 151: 1283-1295Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar Because this model is on the C57BL/6 background and our previous gene transfer experiments were performed on CD1 mice, we tested whether exo-AAV transduces hair cells with the same efficiency on the C57BL/6 background. We did not observe any differences between CD1 and C57BL/6 transduction rates using exo-AAV1-GFP (Figure S5). Although results were variable, we noted that some of the C57BL/6 animals showed very efficient transduction (>95% IHC and >85% OHC transduction; Figure S6), which was never achieved with conventional AAV1. For gene-addition therapy, a mouse-codon-optimized gene encoding LHFPL5 with a hemagglutinin (HA) tag at the N terminus was cloned into an AAV vector backbone under the CBA promoter (Figure S7A). When this exo-AAV1-HA-Lhfpl5 was produced in HEK293T cells, anti-HA immunoblotting of cell lysates revealed bands of the expected molecular weight for LHFPL5 (Figure S7B). Next, we tested whether this construct restores function in cochlear explant cultures from Lhfpl5−/− animals. Exo-AAV1-HA-Lhfpl5 restored FM1-43 loading in explant cultures (indicating the presence of functional mechanotransduction channels) (Figure 4A). In addition, anti-HA labeling was present in hair cell stereocilia (Figure 4B). We quantified average FM1-43 signal in cochlear explants from Lhfpl5+/− mice, Lhfpl5−/− mice, and Lhfpl5−/− mice transduced in culture with two different doses of exo-AAV1-HA-Lhfpl5. The average FM1-43 fluorescence intensity per hair cell in Lhfpl5−/− cochlea was comparable to the background intensity in an area without hair cells. In Lhfpl5−/− cochleas transduced by exo-AAV1-HA-Lhfpl5 vector, FM1-43 intensity was 70% of the Lhfpl5+/− positive control at the highest tested dose (Figure 4C). FM1-43 intensity increased from apex to base in exo-AAV1-HA-Lhfpl5-treated Lhfpl5−/− cultures, a gradient similar to that seen with the GFP reporter (Figure 1D). At all points, cellular FM1-43 intensity levels were significantly higher than in untreated Lhfpl5−/− cultures (Figure 4D). At the base, FM1-43 intensity was as high in exo-AAV1-HA-Lhfpl5-treated Lhfpl5−/− cultures as in heterozygous positive controls (Figure 4D). These data confirmed that the construct was functional and that the HA tag allowed specific detection of the transgene. Next, we injected exo-AAV1-HA-Lhfpl5 into the cochlea by RWM injection at P1 to P2. RWM injection was used rather than cochleostomy because it was less variable in our hands. Furthermore, we could use a higher volume and therefore dose using RWM injection, and there was less of base-to-apex decrease in transduction with RMW injection compared to cochleostomy (Figure 2D). For in vivo injection, we administered the maximum injectable volume based on preliminary experiments: 1,200 nL (containing 2.7 × 109 GCs). Several days later, we dissected cochleas and cultured them for 1 to 2 days before viewing. Anti-HA immunostaining at P4+2 showed distinct signal in stereociliary bundles of both IHCs and OHCs (Figure 5A). High magnification images revealed anti-HA staining at the tips of stereocilia, including the tallest row, in agreement with the previously reported localization of native LHFPL518Xiong W. Grillet N. Elledge H.M. Wagner T.F. Zhao B. Johnson K.R. Kazmierczak P. Müller U. TMHS is an integral component of the mechanotransduction machinery of cochlear hair cells.Cell. 2012; 151: 1283-1295Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar (Figure 5B). We confirmed that exo-AAV-transduced IHCs and OHCs have functional mechanotransduction, as assessed by FM1-43 loading (Figure 5C). We assessed the efficiency of exo-AAV transduction by counting the hair cells with anti-HA labeling at the bundle and found that 72 ± 17% of IHCs and 30 ± 5% of OHCs exhibited bundle staining, with nearly equal distribution along the cochlea (Figure 5D). We also tested AAV-HA-Lhfpl5-internal ribosomal entry site (IRES)-GFP packaged in exo-AAV1. This allows co-expression of LHFPL5 and GFP in the same cell. Importantly, all GFP-positive cells exhibited anti-HA staining, confirming specificity of the anti-HA antibody (Figure S8). Some GFP-negative cells also showed anti-HA bundle staining, which may be due to weak translation downstream of the IRES, making GFP undetectable. To determine whether exo-AAV-mediated gene transfer impairs normal hearing, we tested heterozygous animals injected with exo-AAV1-HA-Lhfpl5 by RWM injection. RWM injection did not alter hearing thresholds, as measured by auditory brainstem evoked responses (ABRs) (Figure 6B) or change ABR P1 or P2 peak amplitudes (Figure 6C), confirming that both the procedure and the vectors are safe at early ages. Next, we tested physiological rescue of hearing in deaf mice injected with exo-AAV1-HA-Lhfpl5 and performed ABR recordings at 4 weeks post-injection using frequencies from 4 to 45 kHz (Figures 6A–6C). Uninjected Lhfpl5−/− animals did not show detectable ABRs at any sound pressure level (SPL) up to 100 dB (Figure 6A). In Lhfpl5−/− animals injected through the RWM with exo-AAV1-HA-Lhfpl5, we observed improved hearing thresholds at frequencies from 4 to 22 kHz (Figure 6B) in 9 out of 12 animals. The four animals with the best rescue showed thresholds of ∼70 dB SPL at 8 and 11 kHz, an improvement of ∼30 dB. We never detected ABRs for sound presented to the non-injected side. Although we did not directly analyze gene transfer in the non-injected ear, the ABR data suggest that any gene transfer to the contralateral ear is minimal using this injection protocol. Nevertheless, we have previously reported low levels of gene transfer to inner ear hair cells after intravenous injection of exo-AAV in adult mice, suggesting that it may be possible to transduce both ears under certain conditions.19Hudry E. Martin C. Gandhi S. György B. Scheffer D.I. Mu D. Merkel S.F. Mingozzi F. Fitzpatrick Z. Dimant H. et al.Exosome-associated AAV vector as a robust and convenient neuroscience tool.Gene Ther. 2016; 23: 380-392Crossref PubMed Scopus (84) Google Scholar The average peak 2 amplitudes at 90 dB SPL were 0.88 ± 0.18 and 0.84 ± 0.12 μV (mean ± SEM) at 8 and 11 kHz, respectively, which is approximately 25% of those in normal heterozygotes (Figure 6C). Latencies of peak 1 and peak 2 were not significantly increased in rescued animals compared to wild-type (WT) animals at the same SPL, except at 11 kHz for peak 1 (Figure 6C; p < 0.01, two-tailed t test). Injected and non-injected heterozygotes did not show a statistically significant difference in the latency of the P1 or P2 ABR peaks (Figure 6C). We tested the behavioral correlates of hearing and balance in Lhfpl5 knockouts (KOs) rescued with exo-AAV. We first found that rescue of hearing by exo-AAV1-HA-Lhfpl5 was sufficient to elicit a startle response to a loud clap, a standard test of hearing (Movie S1). Head bobbing and circling are common traits of Lhfpl5 KO mice and may reflect abnormal vestibular function.20Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. A type VII myosin encoded by the mouse deafness gene shaker-1.Nature. 1995; 374: 62-64Crossref PubMed Scopus (545) Google Scholar, 21Anderson D.W. Probst F.J. Belyantseva I.A. Fridell R.A. Beyer L. Martin D.M. Wu D. Kachar B. Friedman T.B. Raphael Y. et al.The motor and tail regions of myosin XV are critical for normal structure and function of auditory and vestibular hair cells.Hum. Mol. Genet. 2000; 9: 1729-1738Crossref PubMed Scopus (99) Google Scholar, 22Di Palma F. Holme R.H. Bryda E.C. Belyantseva I.A. Pellegrino R. Kachar B. Steel K.P. Noben-Trauth K. Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D.Nat. Genet. 2001; 27: 103-107Crossref PubMed Scopus (366) Google Scholar Because GFP-positive hair cells were also evident in vestibular sensory epithelia, suggesting vector diffusion to the vestibular system (Figure 3), we performed behavioral tests in treated (injected through the RWM with exo-AAV1-HA-Lhfpl5) and nontreated Lhfpl5 KO mice. We performed an open field test, in which animals were placed in a circular arena for 5 min. Normal heterozygous mice showed gait and head stability and normal explorative behavior (Movie S2). On the contrary, Lhfpl5−/− mice exhibited frequent head tossing, gait instability, backward movement, and circling. Five out of 12 exo-AAV1-HA-Lhfpl5-treated Lhfpl5−/− animals did not exhibit head tossing, indicating rescue of balance function. Averaging all animals, we found head tossing was significantly decreased in the treated Lhfpl5−/− animals compared to the untreated animals (Figure 6D) (p < 0.001, Mann-Whitney U test). Similarly, circling was analyzed using computerized image analysis. Treated Lhfpl5−/− animals exhibited significantly fewer 360° rotations compared to untreated animals (Figure 6D) (p < 0.05, Mann-Whitney U test). These results confirm that hearing and the abnormal movements characteristic of compromised balance in these mice are improved after exo-AAV1-HA-Lhfpl5 gene therapy. In this study, we found that exosome-associated AAV transduces cochlear and vestibular hair cells with much greater efficiency than do conventional AAV vectors. Prior studies had shown some transduction of IHCs with conventional AAVs, but little transduction of OHCs.12Akil O. Seal R.P. Burke K. Wang C. Alemi A. During M. Edwards R.H. Lustig L.R. Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.Neuron. 2012; 75: 283-293Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 23Yu Q. Wang Y. Chang Q. Wang J. Gong S. Li H. Lin X. Virally expressed connexin26 restores gap junction function in the cochlea of conditional Gjb2 knockout mice.Gene Ther. 2014; 21: 71-80Crossref PubMed Scopus (82) Google Scholar We found that exo-AAV1 efficiently transduces IHCs and transduces OHCs much more efficiently than does conventional AAV1. AAV1 was our standard of comparison because it has been used in prior studies of hearing gene therapy.12Akil O. Seal R.P. Burke K. Wang C. Alemi A. During M. Edwards R.H. Lustig L.R. Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.Neuron. 2012; 75: 283-293Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 13Askew C. Rochat C. Pan B. Asai Y. Ahmed H. Child E. Schneider B.L. Aebischer P. Holt J.R. Tmc gene therapy restores auditory function in deaf mice.Sci. Transl. Med. 2015; 7: 295ra108Crossref PubMed Scopus (166) Google Scholar In those studies, it was shown to only transduce inner hair cells relatively efficiently. Interestingly, we found that exo-AAV9-GFP was extremely efficient at transduction of cochlear explant cultures, although, in vivo, this serotype performed similarly to exo-AAV1. In order to determine whether the efficient reporter gene levels in HCs achieved with exo-AAV could be translated to expression of HC-relevant genes, we tested exo-AAV-mediated expression of Lhfpl5 in deaf Lhfpl5−/− mice. After RWM injection, we found widespread expression of HA-tagged LHFPL5 throughout the cochlea. Treated Lhfpl5−/− mice were able to respond to sound, as measured physiologically, and showed improvements in balance-related ab" @default.
• W2570201053 created "2017-01-13" @default.
• W2570201053 creator A5006313534 @default.
• W2570201053 creator A5020611876 @default.
• W2570201053 creator A5021938407 @default.
• W2570201053 creator A5039279042 @default.
• W2570201053 creator A5046985587 @default.
• W2570201053 creator A5049104753 @default.
• W2570201053 creator A5057017311 @default.
• W2570201053 creator A5059573074 @default.
• W2570201053 creator A5060352361 @default.
• W2570201053 creator A5064371575 @default.
• W2570201053 creator A5070877004 @default.
• W2570201053 creator A5072413967 @default.
• W2570201053 creator A5079604577 @default.
• W2570201053 creator A5081441104 @default.
• W2570201053 creator A5082147046 @default.
• W2570201053 creator A5085042999 @default.
• W2570201053 date "2017-02-01" @default.
• W2570201053 modified "2023-10-01" @default.
• W2570201053 title "Rescue of Hearing by Gene Delivery to Inner-Ear Hair Cells Using Exosome-Associated AAV" @default.
• W2570201053 cites W1514414732 @default.
• W2570201053 cites W1861297144 @default.
• W2570201053 cites W1963623233 @default.
• W2570201053 cites W1964986281 @default.
• W2570201053 cites W1974078650 @default.
• W2570201053 cites W1978143590 @default.
• W2570201053 cites W1986733224 @default.
• W2570201053 cites W1994710975 @default.
• W2570201053 cites W2004590552 @default.
• W2570201053 cites W2006920577 @default.
• W2570201053 cites W2023266954 @default.
• W2570201053 cites W2023914268 @default.
• W2570201053 cites W2024597974 @default.
• W2570201053 cites W2034060287 @default.
• W2570201053 cites W2041407230 @default.
• W2570201053 cites W2055648999 @default.
• W2570201053 cites W2056602419 @default.
• W2570201053 cites W2060516472 @default.
• W2570201053 cites W2066474908 @default.
• W2570201053 cites W2084189677 @default.
• W2570201053 cites W2088523571 @default.
• W2570201053 cites W2090421229 @default.
• W2570201053 cites W2104183007 @default.
• W2570201053 cites W2131434998 @default.
• W2570201053 cites W2148223197 @default.
• W2570201053 cites W2292311115 @default.
• W2570201053 cites W2317451409 @default.
• W2570201053 cites W3014251530 @default.
• W2570201053 doi "https://doi.org/10.1016/j.ymthe.2016.12.010" @default.
• W2570201053 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5368844" @default.
• W2570201053 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28082074" @default.
• W2570201053 hasPublicationYear "2017" @default.
• W2570201053 type Work @default.
• W2570201053 sameAs 2570201053 @default.
• W2570201053 citedByCount "162" @default.
• W2570201053 countsByYear W25702010532017 @default.
• W2570201053 countsByYear W25702010532018 @default.
• W2570201053 countsByYear W25702010532019 @default.
• W2570201053 countsByYear W25702010532020 @default.
• W2570201053 countsByYear W25702010532021 @default.
• W2570201053 countsByYear W25702010532022 @default.
• W2570201053 countsByYear W25702010532023 @default.
• W2570201053 crossrefType "journal-article" @default.
• W2570201053 hasAuthorship W2570201053A5006313534 @default.
• W2570201053 hasAuthorship W2570201053A5020611876 @default.
• W2570201053 hasAuthorship W2570201053A5021938407 @default.
• W2570201053 hasAuthorship W2570201053A5039279042 @default.
• W2570201053 hasAuthorship W2570201053A5046985587 @default.
• W2570201053 hasAuthorship W2570201053A5049104753 @default.
• W2570201053 hasAuthorship W2570201053A5057017311 @default.
• W2570201053 hasAuthorship W2570201053A5059573074 @default.
• W2570201053 hasAuthorship W2570201053A5060352361 @default.
• W2570201053 hasAuthorship W2570201053A5064371575 @default.
• W2570201053 hasAuthorship W2570201053A5070877004 @default.
• W2570201053 hasAuthorship W2570201053A5072413967 @default.
• W2570201053 hasAuthorship W2570201053A5079604577 @default.
• W2570201053 hasAuthorship W2570201053A5081441104 @default.
• W2570201053 hasAuthorship W2570201053A5082147046 @default.
• W2570201053 hasAuthorship W2570201053A5085042999 @default.
• W2570201053 hasBestOaLocation W25702010531 @default.
• W2570201053 hasConcept C104317684 @default.
• W2570201053 hasConcept C105702510 @default.
• W2570201053 hasConcept C111599444 @default.
• W2570201053 hasConcept C135983454 @default.
• W2570201053 hasConcept C145059251 @default.
• W2570201053 hasConcept C20518536 @default.
• W2570201053 hasConcept C2778500370 @default.
• W2570201053 hasConcept C2781261824 @default.
• W2570201053 hasConcept C54355233 @default.
• W2570201053 hasConcept C86803240 @default.
• W2570201053 hasConcept C95444343 @default.
• W2570201053 hasConceptScore W2570201053C104317684 @default.
• W2570201053 hasConceptScore W2570201053C105702510 @default.
• W2570201053 hasConceptScore W2570201053C111599444 @default.
• W2570201053 hasConceptScore W2570201053C135983454 @default.
• W2570201053 hasConceptScore W2570201053C145059251 @default.
• W2570201053 hasConceptScore W2570201053C20518536 @default.

• next