{"title":"介绍一种新的血管内腺相关病毒介导的基因传递方法。","authors":"Z H Fazal, K Hosaka, F P Manfredsson, B L Hoh","doi":"","DOIUrl":null,"url":null,"abstract":"<p><strong>Introduction: </strong>Adeno-associated virus (AAV) has shown therapeutic potential as a viral vector in various studies of gene therapy. However, research on its use in targeting intravascular cells in a localized manner is lacking. We introduce a novel method to deliver various AAV serotypes intravascularly and examine their efficiency in transducing cells of the murine carotid artery.</p><p><strong>Objective: </strong>The study aimed to examine the transduction efficiency of AAV-mediated gene delivery in cells of the murine carotid artery both with and without a fully-formed aneurysm. Results of infection were visualized with green fluorescence protein (GFP) reporter gene.</p><p><strong>Methods: </strong>Naïve murine carotid artery or experimentally-induced murine carotid aneurysm was ligated distally and proximally. A small incision was made and 5 uL AAV2, AAV5, AAV8, or AAV9 was microsurgically injected and allowed to incubate for 30 min. Incision was closed and tissue was excised three weeks following AAV injection. Carotid artery or aneurysm tissue was excised and fixed in 4% paraformaldehyde solution. On both naïve carotid artery tissue and aneurysm tissue, GFP was visualized by immunofluorescence using antibody against GFP.</p><p><strong>Results: </strong>Three out of four serotypes of AAV successfully transduced cells within both the murine aneurysm tissue and the naïve carotid artery tissue. AAV5- and AAV9-transduced aneurysm tissue showed the greatest presence of GFP, with AAV8 showing less overall fluorescence. AAV2 showed no fluorescence.</p><p><strong>Conclusion: </strong>AAV-mediated gene delivery is an effective way to transduce cells intravascularly with a transgene of interest. Our method can be generalized across a wide variety of studies to further research or treat other vascular disease.</p>","PeriodicalId":92159,"journal":{"name":"Virology (Hyderabad)","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2018-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258074/pdf/","citationCount":"0","resultStr":"{\"title\":\"Introducing a Novel Method of Intravascular Adeno-associated Virus-mediated Gene Delivery.\",\"authors\":\"Z H Fazal, K Hosaka, F P Manfredsson, B L Hoh\",\"doi\":\"\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Introduction: </strong>Adeno-associated virus (AAV) has shown therapeutic potential as a viral vector in various studies of gene therapy. However, research on its use in targeting intravascular cells in a localized manner is lacking. We introduce a novel method to deliver various AAV serotypes intravascularly and examine their efficiency in transducing cells of the murine carotid artery.</p><p><strong>Objective: </strong>The study aimed to examine the transduction efficiency of AAV-mediated gene delivery in cells of the murine carotid artery both with and without a fully-formed aneurysm. Results of infection were visualized with green fluorescence protein (GFP) reporter gene.</p><p><strong>Methods: </strong>Naïve murine carotid artery or experimentally-induced murine carotid aneurysm was ligated distally and proximally. A small incision was made and 5 uL AAV2, AAV5, AAV8, or AAV9 was microsurgically injected and allowed to incubate for 30 min. Incision was closed and tissue was excised three weeks following AAV injection. Carotid artery or aneurysm tissue was excised and fixed in 4% paraformaldehyde solution. On both naïve carotid artery tissue and aneurysm tissue, GFP was visualized by immunofluorescence using antibody against GFP.</p><p><strong>Results: </strong>Three out of four serotypes of AAV successfully transduced cells within both the murine aneurysm tissue and the naïve carotid artery tissue. AAV5- and AAV9-transduced aneurysm tissue showed the greatest presence of GFP, with AAV8 showing less overall fluorescence. AAV2 showed no fluorescence.</p><p><strong>Conclusion: </strong>AAV-mediated gene delivery is an effective way to transduce cells intravascularly with a transgene of interest. 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引用次数: 0
摘要
腺相关病毒(AAV)作为一种病毒载体在各种基因治疗研究中显示出治疗潜力。然而,关于其在局部靶向血管内细胞方面的应用研究尚缺乏。我们介绍了一种新的方法,在血管内传递各种AAV血清型,并检查它们在小鼠颈动脉转导细胞中的效率。目的:本研究旨在检测aav介导的基因传递在小鼠颈动脉细胞中的转导效率,无论是否存在完全形成的动脉瘤。用绿色荧光蛋白(GFP)报告基因显示感染结果。方法:Naïve小鼠颈动脉或实验性小鼠颈动脉瘤远端和近端结扎。切开一个小切口,显微外科注射5 uL AAV2、AAV5、AAV8或AAV9,孵育30分钟。注射AAV后3周,关闭切口,切除组织。切除颈动脉或动脉瘤组织,用4%多聚甲醛溶液固定。在naïve颈动脉组织和动脉瘤组织上,使用抗GFP抗体,用免疫荧光法观察GFP。结果:四种血清型中的三种AAV成功地在小鼠动脉瘤组织和naïve颈动脉组织内转导细胞。AAV5-和aav9转导的动脉瘤组织显示最大的GFP存在,AAV8显示较少的整体荧光。AAV2无荧光。结论:aav介导的基因传递是一种有效的方法,可以在血管内转导细胞。我们的方法可以推广到各种各样的研究,以进一步研究或治疗其他血管疾病。
Introducing a Novel Method of Intravascular Adeno-associated Virus-mediated Gene Delivery.
Introduction: Adeno-associated virus (AAV) has shown therapeutic potential as a viral vector in various studies of gene therapy. However, research on its use in targeting intravascular cells in a localized manner is lacking. We introduce a novel method to deliver various AAV serotypes intravascularly and examine their efficiency in transducing cells of the murine carotid artery.
Objective: The study aimed to examine the transduction efficiency of AAV-mediated gene delivery in cells of the murine carotid artery both with and without a fully-formed aneurysm. Results of infection were visualized with green fluorescence protein (GFP) reporter gene.
Methods: Naïve murine carotid artery or experimentally-induced murine carotid aneurysm was ligated distally and proximally. A small incision was made and 5 uL AAV2, AAV5, AAV8, or AAV9 was microsurgically injected and allowed to incubate for 30 min. Incision was closed and tissue was excised three weeks following AAV injection. Carotid artery or aneurysm tissue was excised and fixed in 4% paraformaldehyde solution. On both naïve carotid artery tissue and aneurysm tissue, GFP was visualized by immunofluorescence using antibody against GFP.
Results: Three out of four serotypes of AAV successfully transduced cells within both the murine aneurysm tissue and the naïve carotid artery tissue. AAV5- and AAV9-transduced aneurysm tissue showed the greatest presence of GFP, with AAV8 showing less overall fluorescence. AAV2 showed no fluorescence.
Conclusion: AAV-mediated gene delivery is an effective way to transduce cells intravascularly with a transgene of interest. Our method can be generalized across a wide variety of studies to further research or treat other vascular disease.