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665. Experiences with Delivery of Nanoparticles to the Mouse Cochlea
NEUROLOGIC: SENSORY Results: The ABR monitoring revealed that there was no loss of the cochlear function over the frequencies tested after AAV vector injection. AAV1-mediated GDNF expression in the cochlea was confirmed by ELISA and immunohistochemistry, and the expression in the cochlear was successfully regulated by the Tet-on system. AAV1-mediated GDNF expression protected the cochlear function from the aminoglycosides-induced ototoxicity in rats. Damaged spiral ganglion cells and hair cells were significantly reduced by GDNF expression as compared to that with EGFP expression. Conclusion: AAV1-mediated GDNF expression preserved the cochlear function from the aminoglycosides-induced ototoxicity in rats. Furthermore, AAV1 vector with the Tet-on system effectively modulates transgene expression in the cochlea. Our results suggested that AAV1-mediated expression of GDNF in the cochlea with regulated transduction system is a promising strategy to protect the cochlea from the aminoglycoside-induced damage.
664. Regeneration of Sensory Cells in the Inner Ear of Deafened Mature Guinea Pigs Donald L. Swiderski,1 Masahiko Izumikawa,1 Douglas E. Brough,2 Yehoash Raphael.1 1 Department of Otolarygology, Kresge Hearing Research Institute, University of Michigan, Ann Arbor, MI; 2Vector Sciences, Research, GenVec Inc, Gaithersburg, MD. The mammalian organ of Corti, the sensory epithelium of the inner ear, is a mosaic composed of sensory cells (hair cells) and nonsensory supporting cells. All cells are highly differentiated in structure and function. Because no undifferentiated cells are held in reserve to replace damaged cells, hearing impairment due to loss of hair cells is permanent. Differentiation of complex tissues like the organ of Corti typically is governed by specific cascades of gene expression. In mice, a critical gene in the differentiation of hair cells is the basic helix-loop-helix transcription factor Atoh1(=Math1), a homolog of the Drosophila gene atonal. Previous studies have shown that inoculating inner ears of normal adult guinea pigs with an adenovirus vector expressing Math1 (Ad.Math1) can generate new cochlear hair cells in regions normally occupied only by supporting cells. In this study, we deafened adult guinea pigs by administration with ototoxic drugs (kanamycin and ethacrynic acid) prior to inoculating their inner ears with Ad.Math1 and evaluated differences in hair cell number and hearing thresholds before and after treatment. Douglas E. Brough is a GenVec employee.
665. Experiences with Delivery of Nanoparticles to the Mouse Cochlea Mark Praetorius,1,2 Kim Baker,2 Bernhard Schick,3 Peter K. Plinkert,1 Hinrich Staecker.2 1 Otolaryngology, Head and Neck Surgery, Ruprecht-KarlsUniversity, Heidelberg, Germany; 2Otolaryngology, Head and Neck Surgery, University of Maryland, Baltimore, MD; 3 Otolaryngology, Head and Neck Surgery, University of ErlangenNuremberg, Erlangen, Germany. Gene transfer in the inner ear has been approached mainly using viral vectors. Recent studies have suggested that gene therapy has the potential to treat sensorineural hearing loss through the transfer of math1. The safety profile of non viral gene therapy makes this form of gene delivery ideal for use in the cochlea. Cochlear non viral gene delivery has been examined by several studies and found to be inefficient compared to viral vector gene transfer. We have examined two separate approaches to non viral gene delivery in the inner ear. We compared the delivery of silica nanoparticles to delivery of a range of delivery of polymer based gene delivery. Two separate
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
approaches to the inner ear were used: Vectors were delivered via application to the round window or delivery into the cochlea via a fenestration of the utricle. Distribution of transgene was assessed in the cochlea as well as in the contralateral ear and in the brain stem. Transfection of the sensory hair cells and the spiral ganglion cells was seen in the cochlear and vestibular organ on both sides, suggesting that the vector spread through the cochlea aqueduct as previously demonstrated in viral vectors. The distal portions of the central auditory pathway (dorsal cochlear nucleus, superior olivary complex) were found to be transfected with the nanoparticles indicating an anterograde axonal transport. Non viral gene transfer using advanced non viral vectors may provide a delivery system to the sensory hair cells and spiral ganglion cells and may even be suited to transfect neurons of the central auditory pathway.
666. LV with Different Promoters Mediated Gene Expression in the Inner Ear Maoli Duan, Anders Fridberger, Dongguang Wei, Cecilia Lundberg. 1 Clinical Neuroscience, Karolinska Hospital, Karolinska Institutet, Stockholm, Sweden; 2Physiological Science, Lund University, Lund, Sweden. Introduction Hearing loss is one of the most common diseases in our society affecting more than 10% of the population. The inner ear is unique organ compared to other system. First, the cochlea is a relatively isolated organ, which minimises unwanted effects on other tissues. The limited direct blood supply of the cochlea should also reduce the risk of immune responses. As the cochlea is fluid-filled, all functionally important cells are easily reached and a wide transfection of the cochlea is expected. These anatomic characteristics provide a beautiful model to carry out gene therapy in the inner ear since we can control the concentration using either a single injection or an osmotic pump to infuse genes into the cochlea chronically. The volume of perilymph and endolymph are charaderised in guinea pig, rat and mouse, the dosage can be controlled tightly and thus the risk of adverse effects a redecreased. Secondly, there is no other peripheral system in our body like the inner ear where so many genes have been cloned during past a few years. Rapid progress has been made in identifying hearing-related genes in the mouse. Thirdly, many transit factors and neurotrophins have been found coupled to development of the inner ear and even to survival of sensory cells of adult’s inner ear against environmental damage such as noise trauma, ototoxic chemicals and ageing. However, we should keep in mind that gene delivery is critical key to make inner ear gene therapy success. At present, the main problem of gene therapy is the lack of efficient, specific and very safe gene delivery system. These bottlenecks are particular in the case for inner ear gene therapy. There is no report to show viral and non-viral vectors can target hair cells in vivo situation according to our knowledge. Thus, we will develop methods to see which vector can target hair cells and cochlear neurons in vivo. Material and Methods We have applied LV with different promoters to drive reporter gene GFP, namely CMV, PKG, CAG and Ef1α to see which promoter is more efficient and specific to target cochlear hair cells and neurons. We inject 1 µl LV with different promoters into the cochlea of rat and mouse and analysed the cochleae using confocal microscopy and immunohistochemical study. Results We found that there is different reporter gene expression in the cochlea using different promoters. There are stronger gene expression in the promoters PKG and CAG but less got less gene expression in Ef1α and no gene expression in CMV.
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664. Regeneration of Sensory Cells in the Inner Ear of Deafened Mature Guinea Pigs Donald L. Swiderski,1 Masahiko Izumikawa,1 Douglas E. Brough,2 Yehoash Raphael.1 1 Department of Otolarygology, Kresge Hearing Research Institute, University of Michigan, Ann Arbor, MI; 2Vector Sciences, Research, GenVec Inc, Gaithersburg, MD. The mammalian organ of Corti, the sensory epithelium of the inner ear, is a mosaic composed of sensory cells (hair cells) and nonsensory supporting cells. All cells are highly differentiated in structure and function. Because no undifferentiated cells are held in reserve to replace damaged cells, hearing impairment due to loss of hair cells is permanent. Differentiation of complex tissues like the organ of Corti typically is governed by specific cascades of gene expression. In mice, a critical gene in the differentiation of hair cells is the basic helix-loop-helix transcription factor Atoh1(=Math1), a homolog of the Drosophila gene atonal. Previous studies have shown that inoculating inner ears of normal adult guinea pigs with an adenovirus vector expressing Math1 (Ad.Math1) can generate new cochlear hair cells in regions normally occupied only by supporting cells. In this study, we deafened adult guinea pigs by administration with ototoxic drugs (kanamycin and ethacrynic acid) prior to inoculating their inner ears with Ad.Math1 and evaluated differences in hair cell number and hearing thresholds before and after treatment. Douglas E. Brough is a GenVec employee.
665. Experiences with Delivery of Nanoparticles to the Mouse Cochlea Mark Praetorius,1,2 Kim Baker,2 Bernhard Schick,3 Peter K. Plinkert,1 Hinrich Staecker.2 1 Otolaryngology, Head and Neck Surgery, Ruprecht-KarlsUniversity, Heidelberg, Germany; 2Otolaryngology, Head and Neck Surgery, University of Maryland, Baltimore, MD; 3 Otolaryngology, Head and Neck Surgery, University of ErlangenNuremberg, Erlangen, Germany. Gene transfer in the inner ear has been approached mainly using viral vectors. Recent studies have suggested that gene therapy has the potential to treat sensorineural hearing loss through the transfer of math1. The safety profile of non viral gene therapy makes this form of gene delivery ideal for use in the cochlea. Cochlear non viral gene delivery has been examined by several studies and found to be inefficient compared to viral vector gene transfer. We have examined two separate approaches to non viral gene delivery in the inner ear. We compared the delivery of silica nanoparticles to delivery of a range of delivery of polymer based gene delivery. Two separate
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
approaches to the inner ear were used: Vectors were delivered via application to the round window or delivery into the cochlea via a fenestration of the utricle. Distribution of transgene was assessed in the cochlea as well as in the contralateral ear and in the brain stem. Transfection of the sensory hair cells and the spiral ganglion cells was seen in the cochlear and vestibular organ on both sides, suggesting that the vector spread through the cochlea aqueduct as previously demonstrated in viral vectors. The distal portions of the central auditory pathway (dorsal cochlear nucleus, superior olivary complex) were found to be transfected with the nanoparticles indicating an anterograde axonal transport. Non viral gene transfer using advanced non viral vectors may provide a delivery system to the sensory hair cells and spiral ganglion cells and may even be suited to transfect neurons of the central auditory pathway.
666. LV with Different Promoters Mediated Gene Expression in the Inner Ear Maoli Duan, Anders Fridberger, Dongguang Wei, Cecilia Lundberg. 1 Clinical Neuroscience, Karolinska Hospital, Karolinska Institutet, Stockholm, Sweden; 2Physiological Science, Lund University, Lund, Sweden. Introduction Hearing loss is one of the most common diseases in our society affecting more than 10% of the population. The inner ear is unique organ compared to other system. First, the cochlea is a relatively isolated organ, which minimises unwanted effects on other tissues. The limited direct blood supply of the cochlea should also reduce the risk of immune responses. As the cochlea is fluid-filled, all functionally important cells are easily reached and a wide transfection of the cochlea is expected. These anatomic characteristics provide a beautiful model to carry out gene therapy in the inner ear since we can control the concentration using either a single injection or an osmotic pump to infuse genes into the cochlea chronically. The volume of perilymph and endolymph are charaderised in guinea pig, rat and mouse, the dosage can be controlled tightly and thus the risk of adverse effects a redecreased. Secondly, there is no other peripheral system in our body like the inner ear where so many genes have been cloned during past a few years. Rapid progress has been made in identifying hearing-related genes in the mouse. Thirdly, many transit factors and neurotrophins have been found coupled to development of the inner ear and even to survival of sensory cells of adult’s inner ear against environmental damage such as noise trauma, ototoxic chemicals and ageing. However, we should keep in mind that gene delivery is critical key to make inner ear gene therapy success. At present, the main problem of gene therapy is the lack of efficient, specific and very safe gene delivery system. These bottlenecks are particular in the case for inner ear gene therapy. There is no report to show viral and non-viral vectors can target hair cells in vivo situation according to our knowledge. Thus, we will develop methods to see which vector can target hair cells and cochlear neurons in vivo. Material and Methods We have applied LV with different promoters to drive reporter gene GFP, namely CMV, PKG, CAG and Ef1α to see which promoter is more efficient and specific to target cochlear hair cells and neurons. We inject 1 µl LV with different promoters into the cochlea of rat and mouse and analysed the cochleae using confocal microscopy and immunohistochemical study. Results We found that there is different reporter gene expression in the cochlea using different promoters. There are stronger gene expression in the promoters PKG and CAG but less got less gene expression in Ef1α and no gene expression in CMV.
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