In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes
In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes
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DOI：
10.1038/sj.gt.3300482
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发表时间：
1997-09-01
期刊：
影响因子：
5.1
通讯作者：
Huang, L
中科院分区：
文献类型：
--
作者：
Li, S;Huang, L
一种用于体内基因转移的新型LPD制剂已经被开发出来。它涉及到质粒DNA与阳离子多肽硫酸鱼精蛋白的相互作用，然后添加DOTAP阳离子脂质体。与DOTAP/DNA复合体相比，LPD能更好地保护质粒DNA免受酶消化，并通过尾静脉注射在小鼠体内持续提供更高的基因表达。当使用荧光素酶报告基因时，被检测的所有组织都有基因表达，包括肺、心、脾、肝和肾，其中肺的表达最高。LPD的体内效率依赖于投料比，也受所用脂类的影响。增加DNA的输送量会导致基因表达的增加。最佳剂量约为每只小鼠50微克，在此浓度下，可在肺中检测到每毫克提取组织蛋白约20纳克的荧光素酶蛋白。将DNA含量增加到每只小鼠100微克，会导致动物的毒性和死亡。肺组织基因表达早在注射后1h即可检测到，6h达高峰，之后逐渐下降。注射后6h，小鼠脾组织中也有高表达，但随后迅速下降。LPD体内表达的基因依赖于给药途径，因为门脉内注射LPD导致肺内基因表达与静脉注射相比下降约100倍。注射。以LacZ为报告基因，结果表明，在肺和脾中，内皮细胞是转基因表达的主要部位。没有发现这些器官有炎症的迹象。由于硫酸鱼精蛋白已被证明是无毒的，对人类只有微弱的免疫原性，这种新的载体可能会用于临床基因治疗。
A novel LPD formulation has been developed for in vivo gene transfer. It involves the interaction of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP/DNA complexes, LPD offers better protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. When a luciferase reporter gene was employed, gene expression was found in all tissues examined including lung, heart, spleen, liver and kidney with the highest expression in the lung. The in vivo efficiency of LPD was dependent upon charge ratio and was also affected by the lipid used. Increasing the amount of DNA delivered induced an increase in gene expression. The optimal dose was approximately 50 mu g per mouse at which concentration approximately 20 ng luciferase protein per milligram extracted tissue protein could be detected in the lung. Increasing the DNA to 100 mu g per mouse resulted in toxicity and death of the animal. Gene expression in the lung was detected as early as 1 h after injection, peaked at 6h and declined thereafter. High expression was also found in the spleen 6 h after injection but dropped very rapidly thereafter. The in vivo gene expressed by LPD was dependent upon the route of administration since intraportal injection of LPD led to about a 100-fold decrease in gene expression in the lung as compared with i.v. injection. Using lacZ as a reporter gene, it was shown that endothelial cells were the primary locus of transgene expression in both the lung and spleen. No sign of inflammation in these organs was noticed. Since protamine sulfate has been proven to be nontoxic and only weakly immunogenic in humans, this novel vector may be useful for clinical gene therapy.