Messenger RNA as a source of transposase for Sleeping Beauty transposon-mediated correction of hereditary tyrosinemia type I
Messenger RNA as a source of transposase for Sleeping Beauty transposon-mediated correction of hereditary tyrosinemia type I
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DOI：
10.1038/sj.mt.6300160
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发表时间：
2007-07-01
影响因子：
12.4
通讯作者：
Wang, Xin
中科院分区：
文献类型：
--
作者：
Wilber, Andrew;Wangensteen, Kirk J.;Wang, Xin
当工程改造的SB转座子与转座酶一起沿着递送时，睡美人（SB）转座子系统介导染色体整合和稳定的基因表达。SB系统的治疗应用中的一个问题是转座酶的持续表达可能导致转座子不稳定性和遗传毒性。在这里，我们测试了使用转座酶编码RNA加转座子DNA校正小鼠延胡索酰乙酰乙酸水解酶（FAH）的缺陷。使用含有小鼠FAH和萤火虫荧光素酶序列的双功能转座子，在递送DNA或RNA作为转座酶来源后，通过体内生物发光成像来跟踪遗传校正的肝组织的生长。以RNA形式提供SB转座酶导致具有FAH和荧光素酶稳定表达的校正肝细胞的选择性再增殖。血浆琥珀酰丙酮和氨基酸水平正常化，表明分解代谢的蛋白质产物的正常肝脏代谢。移植有从初次给药动物分离的肝细胞（250，000）的继发性FAH缺陷动物在2-（2-硝基-4-三氟-甲基苯甲酰基）-1，3-环己二酮（NTBC）停药后存活，体重持续增加，并显示荧光素酶的稳定表达。我们得出结论，转座酶编码的信使RNA（mRNA）可用于介导稳定的非病毒基因治疗，导致完全的表型校正，因此是用于人类基因治疗的重组酶活性的有效来源。
The Sleeping Beauty (SB) transposon system mediates chromosomal integration and stable gene expression when an engineered SB transposon is delivered along with transposase. One concern in the therapeutic application of the SB system is that persistent expression of transposase could result in transposon instability and genotoxicity. Here, we tested the use of transposase-encoding RNA plus transposon DNA for correction of murine fumarylacetoacetate hydrolase (FAH) deficiency. A bi-functional transposon containing both mouse FAH and firefly luciferase sequences was used to track the growth of genetically corrected liver tissue by in vivo bioluminescence imaging after delivery of DNA or RNA as a source of transposase. Supplying SB transposase in the form of RNA resulted in selective repopulation of corrected hepatocytes with stable expression of FAH and luciferase. Plasma succinylacetone and amino acid levels were normalized, suggesting normal liver metabolism of catabolized protein products. Secondary FAH-deficient animals transplanted with hepatocytes (250,000) isolated from primary treated animals survived 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC) withdrawal, gained weight consistently, and demonstrated stable expression of luciferase. We conclude that transposase-encoding messenger RNA (mRNA) can be used to mediate stable non-viral gene therapy, resulting in complete phenotypic correction, and is thus an effective source of recombinase activity for use in human gene therapy.