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Vestibular Hair Cell Regeneration and Re-Establishment of Balance Function Induced by Math1 Gene Delivery
CENTRAL NERVOUS SYSTEM II rhodopsin mRNA. The shRNA-resistant rhodopsin which contains 7 altered amino acid codons (9 altered nucleotides), is expressed at least at the same levels as the wild type rhodopsin according to Western blot analysis and has the same cellular localization as it¹s wild type counterpart as determined by immunofluorescence. Conclusions: A single short hairpin RNA may have application in the treatment of dominant forms of retinal degeneration associated with the entire spectrum of mutations identified in rhodopsin
516. Vestibular Hair Cell Regeneration and ReEstablishment of Balance Function Induced by Math1 Gene Delivery Hinrich Staecker,1 Mark Praetorius,2 Kim Baker,1 Douglas E. Brough.3 1 Otolaryngology Head and Neck Surgery, University of Maryland School of Medicine, Baltimore, MD; 2Otolaryngology, University of Saarland, Homburg, Germany; 3Vector Sciences Research, GenVec Inc, Gaithersburg, MD. Over expression of math1 has been demonstrated to induce generation of hair cells in the cochlea in neonatal tissue culture models of the organ of Corti and math 1 generation of hair cells has recently been shown in vivo. We have examined an in vitro organtypic culture model as well as unilateral acute and bilateral chronic models of aminoglycoside vestibular injury. Delivery of Math1 using an adenovector (Ad) resulted in the generation of hair cells in adult mouse utriclular and saccular cultures. Mice treated acutely or chronically with aminoglycosides showed recovery of a significant portion of the vestibular neuroepithelium after AdMath1.11D was infused into the inner ear. Interestingly there was no recovery of hair cells noted within the cochlea in these mouse models and no hair cells noted outside of the vestibular neuroepithelium. Assessment of animals 2 months after vector infusion demonstrated a recovery of swim times compared to non-vector treated controls, demonstrating recovery of vestibular function after Math1 gene delivery. Doug Brough is an employee of GenVec Inc
517. Prevention of Onset of Parkinson’s Disease by In Vivo Gene Transfer of Human Hepatocyte Growth Factor in Primate Model: A Model of Gene Therapy for Parkinson’s Disease Hiromi Koike,1,2 Akihiko Ishida,3 Munehisa Shimamura,1 Naoyuki Sato,1 Naruya Tomita,1 Yasufumi Kaneda,4 Ryuichi Morishita.1 1 Clinical Gene Therapy Science, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; 2Strategic Drug Discovery, AnGes MG, Inc., Toyonaka, Osaka, Japan; 3Tanabe Central Hospital, Kyotanabe, Kyoto, Japan; 4Gene Therapy Science, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons (DAN) in the substantia nigra (SNi). The ultimate treatment of PD is to prevent DAN from the cell death. We previously showed that hepatocyte growth factor (HGF) resulted in effective prevention of DAN from cell death and remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats. In this study, we have extend the preclinical exploration to primate model of PD. Seven days after stereotaxic transfection of human HGF plasmid or pVAX1 control plasmid into the unilateral striatum, infusion of MPTP into the right internal carotid artery of animals produces toxin-induced injury to the right nigro-striatal pathway with sparing of other dopaminergic neurons on the infused side and with negligible or little injury to the S196
opposite, untreated side. After 2 to 3 weeks period, animals exhibited stable moderate parkinsonian features. But animals transfecterd with human HGF plasmid showed normal states. Interestingly, animals transfected with pVAX1 plasmid demonstrated the apomorphine and amphetamine-induced rotational asymmetry. However, the transfection of human HGF plasmid resulted in a significant inhibition of abnormal rotation over 3 months. In immunohistochemistry, more than 90% doperminergic neurons of PD model animals transfecred with pVAX1 plasmid were lost, while more than 70% those of animals transfected with human HGF plasmid were survived. Microdialysis demonstrated that concentrations of dopamine in striatum and SNi in animals transfected with human HGF plasmid were higher compared with PD model. Overall, the present study demonstrated that over-expression of human HGF prevented neuronal death in primate PD model, providing a potential of novel gene therapy for PD using human HGF plasmid.
518. Long-Term Lentiviral Vector-Mediated Transgene Expression in Neural Progenitor Cells Following Implantation into the Injured Rat Spinal Cord Bas Blits,1 Caroline V. Caperton,1 Brandon M. Kitay,1 Gaelle Corrales,1 Mary Bunge.1 1 The Miami Project to Cure Paralysis, University of Miami, Miami, FL. Due to their self-renewal and multi-potency, stem cells represent an attractive source for cell replacement therapy in neurological disorders. Genetic manipulation of these cells may allow controlled release of therapeutic proteins, suppress immune rejection, or produce essential neurotransmitters. Furthermore, when the expression cassette is incorporated into the host genome ex vivo, this technique also may be used as a method to trace cells following implantation into tissues of interest. We explored the possibility of transducing pluripotent fetal rat cortical neural progenitor cells using lentiviral vectors encoding either the green fluorescent protein (GFP) or neurotrophic factors (NT-3, BDNF, GDNF and CNTF) under control of the CMV promoter and the Woodchuck posttranscriptional regulatory element. Following isolation and expansion of the cells at clonal density on poly-ornithine-fibronectin-coated dishes in the presence of bFGF, cells were collected, infected and replated on the dishes. Staining of these cells for neural markers (such as nestin, GFAP, Tuj-1 and RIP) after transduction did not reveal any significant difference from non-transduced cells. However, when they were transduced with a vector encoding CNTF, cells started expressing GFAP. Cells continued to express the transgene, including when bFGF was withdrawn and when cells started to differentiate into GFAP positive cells. Following delayed (1 week) implantation into the lesion site of the moderately contused spinal cord, transduced cells survived well up to 4 weeks post-implantation (the longest time point currently examined). Migration of the cells was mainly restricted to white matter on either side of the lesion. Currently, the therapeutic and axonal growth stimulating properties of the implanted cells are being investigated in injured rats.
Molecular Therapy Volume 9, Supplement 1, Ma y 2004
Copyright The American Society of Gene Therapy
516. Vestibular Hair Cell Regeneration and ReEstablishment of Balance Function Induced by Math1 Gene Delivery Hinrich Staecker,1 Mark Praetorius,2 Kim Baker,1 Douglas E. Brough.3 1 Otolaryngology Head and Neck Surgery, University of Maryland School of Medicine, Baltimore, MD; 2Otolaryngology, University of Saarland, Homburg, Germany; 3Vector Sciences Research, GenVec Inc, Gaithersburg, MD. Over expression of math1 has been demonstrated to induce generation of hair cells in the cochlea in neonatal tissue culture models of the organ of Corti and math 1 generation of hair cells has recently been shown in vivo. We have examined an in vitro organtypic culture model as well as unilateral acute and bilateral chronic models of aminoglycoside vestibular injury. Delivery of Math1 using an adenovector (Ad) resulted in the generation of hair cells in adult mouse utriclular and saccular cultures. Mice treated acutely or chronically with aminoglycosides showed recovery of a significant portion of the vestibular neuroepithelium after AdMath1.11D was infused into the inner ear. Interestingly there was no recovery of hair cells noted within the cochlea in these mouse models and no hair cells noted outside of the vestibular neuroepithelium. Assessment of animals 2 months after vector infusion demonstrated a recovery of swim times compared to non-vector treated controls, demonstrating recovery of vestibular function after Math1 gene delivery. Doug Brough is an employee of GenVec Inc
517. Prevention of Onset of Parkinson’s Disease by In Vivo Gene Transfer of Human Hepatocyte Growth Factor in Primate Model: A Model of Gene Therapy for Parkinson’s Disease Hiromi Koike,1,2 Akihiko Ishida,3 Munehisa Shimamura,1 Naoyuki Sato,1 Naruya Tomita,1 Yasufumi Kaneda,4 Ryuichi Morishita.1 1 Clinical Gene Therapy Science, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; 2Strategic Drug Discovery, AnGes MG, Inc., Toyonaka, Osaka, Japan; 3Tanabe Central Hospital, Kyotanabe, Kyoto, Japan; 4Gene Therapy Science, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons (DAN) in the substantia nigra (SNi). The ultimate treatment of PD is to prevent DAN from the cell death. We previously showed that hepatocyte growth factor (HGF) resulted in effective prevention of DAN from cell death and remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats. In this study, we have extend the preclinical exploration to primate model of PD. Seven days after stereotaxic transfection of human HGF plasmid or pVAX1 control plasmid into the unilateral striatum, infusion of MPTP into the right internal carotid artery of animals produces toxin-induced injury to the right nigro-striatal pathway with sparing of other dopaminergic neurons on the infused side and with negligible or little injury to the S196
opposite, untreated side. After 2 to 3 weeks period, animals exhibited stable moderate parkinsonian features. But animals transfecterd with human HGF plasmid showed normal states. Interestingly, animals transfected with pVAX1 plasmid demonstrated the apomorphine and amphetamine-induced rotational asymmetry. However, the transfection of human HGF plasmid resulted in a significant inhibition of abnormal rotation over 3 months. In immunohistochemistry, more than 90% doperminergic neurons of PD model animals transfecred with pVAX1 plasmid were lost, while more than 70% those of animals transfected with human HGF plasmid were survived. Microdialysis demonstrated that concentrations of dopamine in striatum and SNi in animals transfected with human HGF plasmid were higher compared with PD model. Overall, the present study demonstrated that over-expression of human HGF prevented neuronal death in primate PD model, providing a potential of novel gene therapy for PD using human HGF plasmid.
518. Long-Term Lentiviral Vector-Mediated Transgene Expression in Neural Progenitor Cells Following Implantation into the Injured Rat Spinal Cord Bas Blits,1 Caroline V. Caperton,1 Brandon M. Kitay,1 Gaelle Corrales,1 Mary Bunge.1 1 The Miami Project to Cure Paralysis, University of Miami, Miami, FL. Due to their self-renewal and multi-potency, stem cells represent an attractive source for cell replacement therapy in neurological disorders. Genetic manipulation of these cells may allow controlled release of therapeutic proteins, suppress immune rejection, or produce essential neurotransmitters. Furthermore, when the expression cassette is incorporated into the host genome ex vivo, this technique also may be used as a method to trace cells following implantation into tissues of interest. We explored the possibility of transducing pluripotent fetal rat cortical neural progenitor cells using lentiviral vectors encoding either the green fluorescent protein (GFP) or neurotrophic factors (NT-3, BDNF, GDNF and CNTF) under control of the CMV promoter and the Woodchuck posttranscriptional regulatory element. Following isolation and expansion of the cells at clonal density on poly-ornithine-fibronectin-coated dishes in the presence of bFGF, cells were collected, infected and replated on the dishes. Staining of these cells for neural markers (such as nestin, GFAP, Tuj-1 and RIP) after transduction did not reveal any significant difference from non-transduced cells. However, when they were transduced with a vector encoding CNTF, cells started expressing GFAP. Cells continued to express the transgene, including when bFGF was withdrawn and when cells started to differentiate into GFAP positive cells. Following delayed (1 week) implantation into the lesion site of the moderately contused spinal cord, transduced cells survived well up to 4 weeks post-implantation (the longest time point currently examined). Migration of the cells was mainly restricted to white matter on either side of the lesion. Currently, the therapeutic and axonal growth stimulating properties of the implanted cells are being investigated in injured rats.
Molecular Therapy Volume 9, Supplement 1, Ma y 2004
Copyright The American Society of Gene Therapy