Specific Targeting And Biological Distribution Of Adeno-Associated Virus Vectors In Inner Ear Delivery

The impressive achievement has been obtained in some mouse models for human deafness through adeno-associated viral(AAV)mediated gene therapy.AAV is one of the most popular viral vectors in gene therapy due to its many advantages,such as low immunogenicity,high safety,broad host range,and long-term stable expression.The effectiveness and safety of gene therapy rely on AAV vectors delivering the transgene into target cells or tissues while minimizing distribution in non-target tissues.Despite the tissue-specific targeting exhibited by various AAVs,there is a lack of specific AAVs capable of infecting particular cell types in inner ear,such as hair cells or supporting cells.Safety concerns have hindered the translation of gene therapy strategies for deafness into clinical trials.Therefore,this thesis explores the transduction of different AAVs in the inner ear and their biodistribution after inner ear delivery,which holds significant importance in transitioning gene therapy for deafness from animal models to clinical translation.Objectives:1.Cellular transduction efficiency and tissue affinity are influenced by different AAVs.To evaluate the transduction efficiency of AAV vectors,including natural serotypes(AAV1,AAV2,AAV9)and novel variants(Anc80L65,AAV-PHP.B,AAVPHP.eB),within the cochlear hair cells(HCs).This assessment is critical for selecting appropriate AAV vectors for gene therapy targeting hearing loss.2.To compare the distribution of non-target organs among different AAV vectors following unilateral RWM administration.3.To assess the transduction efficiency of hair cells(HCs)and their distribution within the brain following the delivery of AAV-PHP.eB into the inner ear through various routes and at different time points.Additionally,we seek to investigate the pathways through which drugs enter the brain from the cochlea.Methods:1 Six AAV subtypes,including AAV1,AAV2,Anc80L65,AAV9,AAV-PHP.B,and AAV-PHP.eB,were injected into the inner ears of neonatal mice(P2-3)using the RWM technique.Cochlear whole mounts from both ears were obtained at P28-30 and subjected to immunofluorescence staining.The samples were then scanned using confocal microscopy to analyze the transduction efficiencies of hair cells(HCs)with different AAV subtypes in both ears2 Six AAV subtypes were administered via the RWM technique to neonatal mice.At P28-30,tissues from the brain,heart,and liver were collected for frozen sectioning and subsequent immunofluorescence staining.Transduction in non-target organs was analyzed using a VS-120 microscope and laser scanning confocal microscopy.Quantitative polymerase chain reaction(Q-PCR)was used to compare the vector genomes between bilateral cochleae and non-target organs such as the brain,heart,and liver.3.ICR wild-type mice were used during both the neonatal period(P2-3)and the adult period(P28-30).AAV-PHP.eB was administered to the inner ear through three different routes: the round window membrane(RWM),the posterior semicircular canal(PSCC),and the utricle.After 3-4 weeks,auditory function was assessed to evaluate the safety of the procedures.Cochlear whole mounts were subjected to immunofluorescence staining and scanned using a confocal microscope to calculate the transduction rate of hair cells(HCs)for different routes and injection timings.Brain sections were prepared for immunofluorescence staining to quantify e GFP-positive cells in various brain regions.Additionally,methylene blue was injected into the inner ear through the RWM,PSCC,and utricle approaches to investigate potential pathways leading to brain transduction.Results:1 AAV1,Anc80L65,AAV9,AAV-PHP.B,and AAV-PHP.eB demonstrated robust IHC transduction rates in the apex,middle,and base turns(93.34-100%).There were no significant differences between these groups.AAV2 showed an IHC transduction rate of about 73.64% in the apex turn(P < 0.05),which was significantly lower than the other groups.There were some variations in OHC transduction efficiency among AAV2,Anc80L65,AAV9,AAV-PHP.B,and AAV-PHP.eB(70.97-97.10%),but these differences were not statistically significant.AAV1 exhibited the lowest OHC transduction efficiency in the apex,middle,and base turns(6.85 ± 5.77%,15.33 ±3.79%,27.50 ± 2.47%),which was significantly lower than the other groups(P <0.0001).We did observe transduction of all six vectors in contralateral ears.In the Anc80L65,AAV-PHP.B,and AAV-PHP.eB groups,the transduction rates in contralateral ears were 50-80% for IHCs and 20-40% for OHCs.2 The e GFP-positive cells of Anc80L65,AAV-PHP.B,and AAV-PHP.eB in the cortex and hippocampus were significantly higher than those of AAV1,AAV2,and AAV9.However,AAV2 consistently demonstrated brighter e GFP expression in the skull base region than the other five serotypes.Limited e GFP signals were found in the hearts and livers among the six AAVs.The vector genomes were dramatically and significantly higher in the injected cochlea than in the contralateral ear,brain,heart,and liver(P < 0.05).The distribution of AAV1 in non-target organs was the lowest compared to the other groups,and AAV-PHP.eB showed the lowest vectors in non-target organs among the three novel variants3 AAV-PHP.eB delivered to newborn mice by RWM,PSCC,and Utricle routes infected cochlear hair cells with high efficiency.The IHC transduction rates were 93.33-100%,and the OHC transduction rates were 88.89-94.44%.When AAV-PHP.eB was injected into adult mouse inner ears using three approaches,the infection efficiencies were 80-100% for IHCs,and less than 10% for OHCs.The three surgical approaches resulted in comparable infection efficiencies.AAV-PHP.eB delivered into the inner ear of adult mice showed equal competence at infecting the IHCs,but the OHC transduction rate(P < 0.0001),mean fluorescence intensity of cochlear whole mounts(P < 0.01),and the e GFP-positive cells in most brain regions(P < 0.05)were significantly lower compared to neonatal period injection.Methylene blue was observed to overflow from the cochlea via the cochlear aqueduct in all three approaches Conclusions:1.Anc80L65,AAV9,AAV-PHP.B,and AAV-PHP.eB transduced both IHCs and OHCs with high efficiencies in neonatal mice.AAV2 displayed relatively lower transduction efficiency in apical turn IHCs,and AAV1 exhibited low transduction efficiency in OHCs.These findings indicate that the transduction rates of HCs vary among AAV vectors.Appropriate AAV vectors could be selected based on the ability of HCs tropism in inner ear gene therapy.2.The AAV vectors exhibited varying transduction efficiencies in the brain and peripheral organs.AAV1 had the lowest overall transduction in non-target organs,while AAV-PHP.eB had the lowest transduction among the novel variants compared to levels in the injected ear.Gene therapy should thus consider not only therapeutic effects at target sites but also potential off-target transduction.3.AAV-PHP.eB transducts both IHCs and OHCs with high efficiency in different delivery routes.However,AAV-PHP.eB injected in adult mice showed low capability in transducting cochleas and brain regions compared to neonatal period injection.These observations are crucial for determining the optimal timing of treatment and for assessing safety in clinical trials of inner ear gene therapy.Additionally,the role of the cochlear aqueduct as a significant channel between the inner ear and cranial space deserves further investigation.