携带CAR启动子的重组AAV9病毒在小鼠体内表达分布特性研究
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摘要
基因治疗正日益成为罕见病乃至单基因遗传病的首选治疗策略,提升基因药物的体内表达是重要研究领域。本文选择4种短内含子构建了4种嵌合型启动子CAR、CAS、CAP、CAT,通过Gluc报告基因表达活性比较发现,CAR强度明显高于CA,仅次于CAG。选择CAR启动子构建和制备了携带EGFP报告基因的重组病毒rAAV9-CAR-EGFP,经SDS-PAGE分析可见病毒外壳蛋白条带清晰,表明纯度良好;Southern杂交检测可见基因组以自身互补的双链DNA为主。在新生小鼠和成年小鼠中用静脉注射(IV)和侧脑室注射(ICV)两种途径给药,研究了rAAV9-CAR-EGFP的体内表达分布特性。结果表明,新生鼠ICV注射rAAV9-CAR-EGFP在其脑部和脊髓可以观察到EGFP高转导效率和高强度表达,新生鼠IV注射rAAV9-CAR-EGFP可在脑组织观察到明确的EGFP绿色荧光,表明该病毒可有效穿越血脑屏障,但IV注射对于中枢神经系统的转导效率和表达强度明显低于ICV注射。无论是IV注射还是ICV注射,神经和肌肉系统中都可观察到EGFP的持续表达;同时还可以观察EGFP蛋白在外周组织中广泛分布,其中以肝脏、骨骼肌和心肌最为显著。另外,成年鼠IV注射rAAV9-CAR-EGFP可在肝脏观察到EGFP持续表达,而新生鼠IV或ICV注射则肝脏中EGFP的表达会随动物生长而明显衰减。这些研究结果为我们下一步用AAV9携带CAR启动子介导目的基因表达治疗中枢神经系统疾病如脊髓性肌萎缩症的基因药物设计奠定了基础。
Gene therapy is becoming the ideal choice for monogenic genetic diseases,enhancing the expression of gene drugs is an important research filed. We constructed four chimeric promoters consisting of a CA basic promoter and short intron named CAR,CAS,CAP,CAT. Gluc activity driven by these four chimeric promoters was measured and only the CAR promoter increased Gluc activity significantly compared with that of the CA promoter,and this increase was slightly lower than that of the CAG promoter. We selected the CAR promoter to produce rAAV9-CAR-EGFP. Three capsid proteins,VP1,VP2,and VP3,were shown by sodium dodecyl sulfate – polyacrylamide gel electrophoresis,which demonstrated the high purity of the virus preparations. Southern Blot showed that the virus consisted mainly of self-complementary(sc)DNA with good integrity. Also,we studied the distribution of rAAV9-CAR-EGFP expression in neonatal and adult mice by intravenous(IV)or intracerebroventricular(ICV)injections. Both administration routes could increase green fluorescence in the central nervous system(CNS)and skeletal muscle. ICV injection of rAAV9-CAR-EGFP could cause phenomenal EGFP transduction in the whole brain and spinal cord compared with that by IV injection,and showed that AAV9 could cross the blood – brain barrier. The IV injection route was more efficient in peripheral organs,especially in skeletal muscle,the heart and liver. In addition,IV injection in adult mice could achieve continuous and strong expression in the liver. However,both injection routes in neonatal mice appeared to decrease transduction and fluorescence significantly in the liver. These findings suggest that gene therapy for CNS diseases(e. g.,spinal muscular atrophy) with expression of AAV9-carrying CAR promoter-mediated genes could be attempted.
Gene therapy is becoming the ideal choice for monogenic genetic diseases,enhancing the expression of gene drugs is an important research filed. We constructed four chimeric promoters consisting of a CA basic promoter and short intron named CAR,CAS,CAP,CAT. Gluc activity driven by these four chimeric promoters was measured and only the CAR promoter increased Gluc activity significantly compared with that of the CA promoter,and this increase was slightly lower than that of the CAG promoter. We selected the CAR promoter to produce rAAV9-CAR-EGFP. Three capsid proteins,VP1,VP2,and VP3,were shown by sodium dodecyl sulfate – polyacrylamide gel electrophoresis,which demonstrated the high purity of the virus preparations. Southern Blot showed that the virus consisted mainly of self-complementary(sc)DNA with good integrity. Also,we studied the distribution of rAAV9-CAR-EGFP expression in neonatal and adult mice by intravenous(IV)or intracerebroventricular(ICV)injections. Both administration routes could increase green fluorescence in the central nervous system(CNS)and skeletal muscle. ICV injection of rAAV9-CAR-EGFP could cause phenomenal EGFP transduction in the whole brain and spinal cord compared with that by IV injection,and showed that AAV9 could cross the blood – brain barrier. The IV injection route was more efficient in peripheral organs,especially in skeletal muscle,the heart and liver. In addition,IV injection in adult mice could achieve continuous and strong expression in the liver. However,both injection routes in neonatal mice appeared to decrease transduction and fluorescence significantly in the liver. These findings suggest that gene therapy for CNS diseases(e. g.,spinal muscular atrophy) with expression of AAV9-carrying CAR promoter-mediated genes could be attempted.
引文
[1]Wang D,Tai P W L,Gao G.Adeno-associated virus vector as a platform for gene therapy delivery[J].Nat Rev Drug Discov,2019,18(5):358-378.
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[3]Deverman B E,Pravdo P L,Simpson B P,Kumar SR,Chan K Y,Banerjee A,Wu W L,Yang B,Huber N,Pasca S P,Gradinaru V.Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain[J].Nat Biotechnol,2016,34(2):204-209.
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[9]Nicole Armbruster,Annalisa Lattanzi,Matthieu Jeavons,Laetitia Van Wittenberghe,Bernard Gjata,Thibaut Marais,Samia Martin,Alban Vignaud,Thomas Voit,Fulvio Mavilio,Martine Barkats and Ana Buj-Bello.Nicole Armbruster,Annalisa Lattanzi,Matthieu Jeavons,Laetitia Van Wittenberghe,Bernard Gjata,Thibaut Marais,Samia Martin,Alban Vignaud,Thomas Voit,Fulvio Mavilio,Martine Barkats,Ana Buj-Bello.Efficacy and biodistribution analysis of intracerebroventricular administration of an optimized scAAV9-SMN1 vector in a mouse model of spinal muscular atrophy[J].Mol Ther Methods Clin Dev,2016,3:16060.
[10]Armbruster N,Lattanzi A,Jeavons M,Van Wittenberghe L,Gjata B,Marais T,Martin S,Vignaud A,Voit T,Mavilio F,Barkats M,Buj-Bello A[J].Mol Ther Methods Clin Dev,2015,23(3):477-487.
[11]Mendell J R,Al-Zaidy S,Shell R,Arnold W D,Rodino-Klapac L R,Prior T W,Lowes L,Alfano L,Berry K,Church K,Kissel J T,Nagendran S,L′Italien J,Sproule D M,Wells C,Cardenas J A,Heitzer M D,Kaspar A,Corcoran S,Braun L,Likhite S,Miranda C,Meyer K,Foust KD,Burghes A H M,Kaspar B K.Single-dose gene-replacement therapy for spinal muscular atrophy.[J].N Engl J Med,2017,377(18):1713-1722.
[2]Chan K Y,Jang M J,Yoo B B,Greenbaum A,Ravi N,Wu W L,Sánchez-Guardado L,Lois C,Mazmanian S K,Deverman B E,Gradinaru V.Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems[J].Nat Neurosci,2017,20(8):1172-1179.
[3]Deverman B E,Pravdo P L,Simpson B P,Kumar SR,Chan K Y,Banerjee A,Wu W L,Yang B,Huber N,Pasca S P,Gradinaru V.Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain[J].Nat Biotechnol,2016,34(2):204-209.
[4]Foust K D,Nurre E,Montgomery C L,Hernandez A,Chan C M,Kaspar B K.Intravascular AAV9preferentially targets neonatal neurons and adult astrocytes[J].Nat Biotechnol,2009,27(1):59-65.
[5]Collaco R F,Cao X,Trempe J P.A helper virus-free packaging system for recombinant adeno-associated virus vectors[J].Gene(Amsterdam),1999,238(2):397-405.
[6]吴小兵,董小岩,伍志坚,屈建国,侯云德.一种快速高效分离和纯化重组腺病毒伴随病毒载体的方法[J].科学通报,2000,45(19):2071.
[7]Grieger J C,Samulski R J.Packaging capacity of adenoassociated virus serotypes:impact of larger genomes on infectivity and postentry steps[J].J Virol,2005,79(15):9933-9944.
[8]Grieger J C,Choi V W,Samulski R J.Production and characterization of adeno-associated viral vectors[J].Nature Protocols,2006,1(3):1412-1428.
[9]Nicole Armbruster,Annalisa Lattanzi,Matthieu Jeavons,Laetitia Van Wittenberghe,Bernard Gjata,Thibaut Marais,Samia Martin,Alban Vignaud,Thomas Voit,Fulvio Mavilio,Martine Barkats and Ana Buj-Bello.Nicole Armbruster,Annalisa Lattanzi,Matthieu Jeavons,Laetitia Van Wittenberghe,Bernard Gjata,Thibaut Marais,Samia Martin,Alban Vignaud,Thomas Voit,Fulvio Mavilio,Martine Barkats,Ana Buj-Bello.Efficacy and biodistribution analysis of intracerebroventricular administration of an optimized scAAV9-SMN1 vector in a mouse model of spinal muscular atrophy[J].Mol Ther Methods Clin Dev,2016,3:16060.
[10]Armbruster N,Lattanzi A,Jeavons M,Van Wittenberghe L,Gjata B,Marais T,Martin S,Vignaud A,Voit T,Mavilio F,Barkats M,Buj-Bello A[J].Mol Ther Methods Clin Dev,2015,23(3):477-487.
[11]Mendell J R,Al-Zaidy S,Shell R,Arnold W D,Rodino-Klapac L R,Prior T W,Lowes L,Alfano L,Berry K,Church K,Kissel J T,Nagendran S,L′Italien J,Sproule D M,Wells C,Cardenas J A,Heitzer M D,Kaspar A,Corcoran S,Braun L,Likhite S,Miranda C,Meyer K,Foust KD,Burghes A H M,Kaspar B K.Single-dose gene-replacement therapy for spinal muscular atrophy.[J].N Engl J Med,2017,377(18):1713-1722.