Ultrasound Directs a Transposase System for Durable Hepatic Gene Delivery in Mice

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• 作者：Cynthia D. Anderson ; Johann Urschitz ; Mark Khemmani ; Jesse B. Owens ; Stefan Moisyadi ; Ralph V. Shohet ; Chad B. Walton
• 关键词：Ultrasound ; Microbubbles ; Hepatic gene therapy ; Transposase ; Site-specific chromosomal integration
• 刊名：Ultrasound in Medicine and Biology
• 出版年：December, 2013
• 年：2013
• 卷：39
• 期：12
• 页码：2351-2361
• 全文大小：1214 K

文摘

Our aim was to evaluate the delivery of transposase-based vectors by ultrasound targeted microbubble destruction (UTMD) in mice. DNA vectors were attached to cationic lipid microbubbles (1-3聽渭m in diameter), injected intravenously and delivered to the liver by destruction of the carrier bubbles with ultrasound in burst mode at 1.0聽MHz, 20-渭s pulse duration, 10-Hz pulse repetition frequency and 鈭?.3-MPa acoustic peak negative pressure. We evaluated the expression and genomic integration of conventional (pcDNA3) and piggyBac transposase-based (pmGENIE) reporter vectors. In聽vivo, we observed UTMD-mediated liver-specific expression of pmGENIE for an average of 24聽d, compared with 4聽d with pcDNA3. Reporter expression was located predominately near blood vessels initially, whereas expression after 3聽d was more evenly distributed through the parenchyma of the liver. We confirmed random genomic integration for pmGENIE in聽vitro; however, integration events for pmGENIE in聽vivo were targeted to specific areas of chromosome 14. Our results suggest that a combination of UTMD and non-viral DNA transposase vectors can mediate weeks of hepatic-specific gene transfer in聽vivo, and analyses performed by non-restrictive linear amplification-mediated (nrLAM) polymerase chain reaction, cloning and sequencing identify an unexpected tropism for integration within a specific sequence on chromosome 14 in聽mice. UTMD delivery of transgenes may be useful for the treatment of hepatic gene deficiency disorders.