Delivery of unmodified bioactive ribozymes by an RNA-stabilizing polyethylenimine (LMW-PEI) efficiently down-regulates gene expression
Delivery of unmodified bioactive ribozymes by an RNA-stabilizing polyethylenimine (LMW-PEI) efficiently down-regulates gene expression
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DOI：
10.1038/sj.gt.3301839
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发表时间：
2002-12-01
期刊：
影响因子：
5.1
通讯作者：
Czubayko, F
中科院分区：
文献类型：
--
作者：
Aigner, A;Fischer, D;Czubayko, F
通过核酶靶向的RNA分子的序列特异性切割是特别有吸引力的，因为它允许有效废除蛋白质表达。然而，到目前为止，在没有化学修饰的情况下，酶活性RNA分子（核酶）的使用受到核酶不稳定性和细胞摄取差的严重阻碍。在本文中，我们提出了一种保护和细胞输送的核酶与低分子量聚乙烯亚胺（LMW-PEI）的复合方法。我们表明，LMW-PEI几乎完全稳定核酶或任何RNA在体外降解。在它们的高效细胞摄取后，无毒的LMW-PEI复合的核酶在低浓度下已经显示出细胞内生物活性，如通过不同细胞系中两种不同基因的下调所证明的。在体内，LMW-PEI复合的核酶在腹膜内（i. p.）注射，显示延长的循环时间，并且在皮下（s.c.）注射后60分钟的肿瘤块。此外，腹膜内注射靶向生长因子多效生长因子（PTN）的LMW-PEI复合核酶导致s.c.人黑色素瘤肿瘤生长和肿瘤内PTN水平。因此，本文描述了一种新的方法，在体外和体内的任何生物活性的RNA核酶的外源性输送没有化学修饰。
The sequence-specific cleavage of RNA molecules through ribozyme targeting is particularly attractive since it allows the effective abrogation of protein expression. So far, however, use of enzymatically active RNA molecules (ribozymes) has, without chemical modification, been severely hampered by ribozyme instability and poor cellular uptake. In this paper, we present a method for protection and cellular delivery of ribozymes by complexation with a low molecular weight polyethylenimine (LMW-PEI). We show that LMW-PEI almost completely stabilizes ribozymes or any RNA against degradation in vitro. Upon their highly efficient cellular uptake, non-toxic LMW-PEI-complexed ribozymes display intracellular bioactivity already at low concentrations as demonstrated by down-regulation of two different genes in different cell lines. In vivo, LMW-PEI-complexed ribozymes were stabilized after intraperitoneal (i.p.) injections, showed prolonged circulation time and intact ribozymes were detected in the subcutaneous (s.c.) tumor mass 60 min after the injection. In addition, i.p. injections of LMW-PEI-complexed ribozymes targeted against the growth factor pleiotrophin (PTN) resulted in marked reduction of s.c. human melanoma tumor growth and of intratumoral PTN levels in a mouse xenograft model. Thus, this paper describes a novel method for exogenous delivery of any bioactive RNA ribozyme in vitro and in vivo without chemical modification.