The use of gene therapy has been intensively studied as a potential method to treat Parkinson’s disease (PD) and other degenerative brain diseases. However, the effects of experimental measures and approaches on the ...The use of gene therapy has been intensively studied as a potential method to treat Parkinson’s disease (PD) and other degenerative brain diseases. However, the effects of experimental measures and approaches on the outcome of gene delivery or on the physiological state of target tissues have not been analyzed as much and systematically. Therefore, we have infused adenovirus vectors expressing either a therapeutic tyrosine hydroxylase (TH) gene or a lacZ reporter gene into striatum in a rat model of PD. The experimental procedures were tested using the Ad lacZ vector in order to optimize concentrations, volumes, infusion speeds and transfection times. The expression of Ad lacZ vector was lower and declined earlier in the lesioned than unlesioned striatum suggesting that the lesion affects on the transfection efficiency and outcome of gene transfection. The effect of three different approaches of Ad TH vector transfection was compared: 1) the delivery of Ad TH gene vector alone into one single site of striatum, 2) the delivery of Ad TH gene vector alone into multiple sites of striatum, and 3) the delivery of Ad TH gene vector into one site of striatum followed by a continuous infusion of tetrahydrobiopterin (BH4) cofactor with a mini pump. There was a small and transient unsignificant decrease in the turning behavior when the Ad TH vector was delivered into one site of the striatum. Simultaneous infusion into several sites or together with BH4 cofactor did not improve more the effect of gene delivery. Thus, although the effects were unsignificant, the Ad TH transfection seemed to decrease the turning behavior in the rat model of PD and the optimal effect was seen at some specific doses and time points. Furthermore, the outcome of gene therapy could depend in addition to the amount and efficacy of gene vectors also on the physiological state and experimental strategies.展开更多
BACKGROUND：Gene therapy for Parkinson＇s disease is being explored as an effective strategy to restore and protect the function of neuronal cells in the substantia nigra. Regulation of gene expression is necessary fo...BACKGROUND：Gene therapy for Parkinson＇s disease is being explored as an effective strategy to restore and protect the function of neuronal cells in the substantia nigra. Regulation of gene expression is necessary for gene therapy to avoid adverse effects due to excessive synthesis of transgene products.OBJECTIVE：Here we developed recombinant adeno-associated virus （AAV） as a viral vector-mediated gene regulation system based on Cre recombinase fused to the mutated ligand-binding domain of the estrogen receptor （CreERT2） ＋ inducing agent tamoxifen. Inducible Cre recombinase was used to reduce tyrosine hydroxylase gene expression and to prevent the excessive increase in dopamine.DESIGN, TIME AND SETTING：A genetic engineering in vitro comparative study and randomized controlled animal experiment. This study was conducted at the Gene Therapy Center, Jichi Medical School, Japan from June 2002 to June 2004.METHODS：To construct a recombinant AAV vector carrying a dopamine synthase gene. The tyrosine hydroxylase gene was inserted using a IoxP fragment that could be regulated by Cre recombinase. The recombinant AAV vector carrying the CreERT2 gene was co-transduced with HEK293 cells and the corpus striatum in a rat model of Parkinson＇s disease, with inducing agent tamoxifen to regulate gene expression.MAIN OUTCOME MEASURES：The levels of dopamine and aromatic L-amino acid decarboxylase （AADC） activity were detected in HEK293 cell medium and in the corpus striatum in a rat model of Parkinson＇s disease using high-performance liquid chromatography. Immunofluorescence double staining was used to observe tyrosine hydroxylase and Cre or AADC co-expression in HEK293 cell medium. Immunohistochemical staining was employed to observe tyrosine hydroxylase and AADC expression and behavioral changes were measured in Parkinson＇s rats.RESULTS：Transfected AAV-CreERT2 and AAV expressing dopamine synthesis enzymes could increase the synthesis of dopamine in HEK293 medium and Parkinson＇s rat striatum （P 〈 0.01） and improve the rotational behavior of Parkinson＇s rats. While tamoxifen markedly reduced overproduction of dopamine caused by cotransfection of viral vectors （P 〈 0.01）, but did not affect the expression and activity of AADC.CONCLUSION：The application of AAV vector-encoded tyrosine hydroxylase gene under the gene regulation system of Cre-ERT2〉, after tamoxifen treatment, can effectively control the generation of genetically modified products to reduce the production of excessive dopamine in vivo and in vitro. Therefore, this method can increase the safety of gene therapy.展开更多
文摘The use of gene therapy has been intensively studied as a potential method to treat Parkinson’s disease (PD) and other degenerative brain diseases. However, the effects of experimental measures and approaches on the outcome of gene delivery or on the physiological state of target tissues have not been analyzed as much and systematically. Therefore, we have infused adenovirus vectors expressing either a therapeutic tyrosine hydroxylase (TH) gene or a lacZ reporter gene into striatum in a rat model of PD. The experimental procedures were tested using the Ad lacZ vector in order to optimize concentrations, volumes, infusion speeds and transfection times. The expression of Ad lacZ vector was lower and declined earlier in the lesioned than unlesioned striatum suggesting that the lesion affects on the transfection efficiency and outcome of gene transfection. The effect of three different approaches of Ad TH vector transfection was compared: 1) the delivery of Ad TH gene vector alone into one single site of striatum, 2) the delivery of Ad TH gene vector alone into multiple sites of striatum, and 3) the delivery of Ad TH gene vector into one site of striatum followed by a continuous infusion of tetrahydrobiopterin (BH4) cofactor with a mini pump. There was a small and transient unsignificant decrease in the turning behavior when the Ad TH vector was delivered into one site of the striatum. Simultaneous infusion into several sites or together with BH4 cofactor did not improve more the effect of gene delivery. Thus, although the effects were unsignificant, the Ad TH transfection seemed to decrease the turning behavior in the rat model of PD and the optimal effect was seen at some specific doses and time points. Furthermore, the outcome of gene therapy could depend in addition to the amount and efficacy of gene vectors also on the physiological state and experimental strategies.
基金Supported by Grants from Ministry of Education,Sci-ence,Sports and Culture,the Japanese Government,and from the Japan Ministry of Health,Labour and WelfareFunds from the Cell Science Research Foundation
文摘BACKGROUND：Gene therapy for Parkinson＇s disease is being explored as an effective strategy to restore and protect the function of neuronal cells in the substantia nigra. Regulation of gene expression is necessary for gene therapy to avoid adverse effects due to excessive synthesis of transgene products.OBJECTIVE：Here we developed recombinant adeno-associated virus （AAV） as a viral vector-mediated gene regulation system based on Cre recombinase fused to the mutated ligand-binding domain of the estrogen receptor （CreERT2） ＋ inducing agent tamoxifen. Inducible Cre recombinase was used to reduce tyrosine hydroxylase gene expression and to prevent the excessive increase in dopamine.DESIGN, TIME AND SETTING：A genetic engineering in vitro comparative study and randomized controlled animal experiment. This study was conducted at the Gene Therapy Center, Jichi Medical School, Japan from June 2002 to June 2004.METHODS：To construct a recombinant AAV vector carrying a dopamine synthase gene. The tyrosine hydroxylase gene was inserted using a IoxP fragment that could be regulated by Cre recombinase. The recombinant AAV vector carrying the CreERT2 gene was co-transduced with HEK293 cells and the corpus striatum in a rat model of Parkinson＇s disease, with inducing agent tamoxifen to regulate gene expression.MAIN OUTCOME MEASURES：The levels of dopamine and aromatic L-amino acid decarboxylase （AADC） activity were detected in HEK293 cell medium and in the corpus striatum in a rat model of Parkinson＇s disease using high-performance liquid chromatography. Immunofluorescence double staining was used to observe tyrosine hydroxylase and Cre or AADC co-expression in HEK293 cell medium. Immunohistochemical staining was employed to observe tyrosine hydroxylase and AADC expression and behavioral changes were measured in Parkinson＇s rats.RESULTS：Transfected AAV-CreERT2 and AAV expressing dopamine synthesis enzymes could increase the synthesis of dopamine in HEK293 medium and Parkinson＇s rat striatum （P 〈 0.01） and improve the rotational behavior of Parkinson＇s rats. While tamoxifen markedly reduced overproduction of dopamine caused by cotransfection of viral vectors （P 〈 0.01）, but did not affect the expression and activity of AADC.CONCLUSION：The application of AAV vector-encoded tyrosine hydroxylase gene under the gene regulation system of Cre-ERT2〉, after tamoxifen treatment, can effectively control the generation of genetically modified products to reduce the production of excessive dopamine in vivo and in vitro. Therefore, this method can increase the safety of gene therapy.
