Defining strategies to extend duration of gene expression from targeted compacted DNA vectors
Defining strategies to extend duration of gene expression from targeted compacted DNA vectors
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DOI：
10.1038/sj.gt.3302299
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发表时间：
2004-09-01
期刊：
影响因子：
5.1
通讯作者：
Davis, PB
中科院分区：
文献类型：
--
作者：
Ziady, AG;Kim, J;Davis, PB
含有聚 L-赖氨酸 (poly-K) 和 DNA 的基因转移复合物以及针对丝氨酸蛋白酶抑制剂复合物受体 (sec-R) 的配体将报告基因传递到体内受体细胞。当使用含有长链聚钾的复合物时，表达持续约 30 天。如果纠正遗传缺陷是目标，那么延长表达持续时间将是可取的。为了测试表达消失的机制是否是由于对转基因的免疫反应或转基因的丢失，我们进行了两项实验。首先，我们将 sec-R 靶向的 lacZ 复合物静脉注射到经过基因工程改造的小鼠体内，以在发育过程中短暂表达该基因。这些应该将这种蛋白质识别为“自身”的小鼠在 30 天后也消除了 lacZ 的表达。在第二个实验中，我们给免疫缺陷动物注射了 sec-R 靶向的人类因子 IX 复合物。在 Rag-1-/- 小鼠中观察到类似的表达时间模式，其中表达也在 40 天后消失。此外，注射复合物50天后，在肺和脾中检测到因子IX质粒DNA，这表明并非所有接受转基因的细胞都被破坏。因此，宿主对转基因的免疫反应可能无法解释这些分子缀合物报告基因表达的丧失。我们进一步测试了 sec-R 靶向复合物的重复施用是否会受到宿主免疫反应的限制。在 40 天内给小鼠预给药两次包含 lacZ 的 sec-R 靶向复合物。然后我们给动物注射静脉注射。使用 sec-R 靶向的人类因子 IX 复合物并测量基因表达和抗体产生。尽管 36 只动物中有 14 只表现出针对目标复合物中配体的低滴度抗体，但与原始剂量相比，表达水平未受影响。当复合物经鼻内给药 3 次（n = 10）时，在血液中未检测到针对该复合物的抗体。来自给予盐水、非靶向复合物或裸DNA的小鼠的血浆不与配体、配体-聚K缀合物或靶向复合物反应。所有表现出人类因子 IX 表达的动物在 21 天时都产生了针对该转基因的抗体。因此，至少三次重复施用sec-R导向的分子缀合物是可能的，只要对转基因本身的免疫反应不受限制。
Gene transfer complexes containing poly-L-lysine (poly-K) and DNA with ligands directed at the serpin enzyme complex receptor (sec-R) deliver reporter genes to receptor-bearing cells in vivo. Expression lasts for about 30 days, when complexes containing long-chain poly-K are used. Extending the duration of expression would be desirable if correction of genetic defects is the goal. To test whether the mechanism by which expression is extinguished was due to an immune response to the transgene, or the loss of the transgene, we conducted two experiments. In the first, we injected sec-R-targeted lacZ complexes intravenously (i.v.) into mice genetically engineered to express this gene briefly during development. These mice, who should recognize the protein as 'self', also extinguished lacZ expression after 30 days. In a second experiment, we injected immunodeficient animals with sec-R-targeted human factor IX complexes. A similar temporal pattern of expression was observed in Rag-1-/- mice, in whom expression also extinguished by 40 days. Moreover, factor IX plasmid DNA was detected in the lung and spleen 50 days after injection of complexes, suggesting that not all cells which had taken up the transgene had been destroyed. Thus, the host's immune response to the transgene may not account for the loss of reporter gene expression from these molecular conjugates. We further tested whether repeat administration of sec-R-targeted complexes will be limited by host immune responses. Mice were pre-dosed twice with sec-R-targeted complexes containing lacZ over a 40-day period. We then injected the animals i.v. with sec-R-targeted human factor IX complexes and measured gene expression and antibody production. Although 14 of 36 animals displayed low-titer antibodies to the ligand in targeted complex, expression levels were unaffected compared with virgin dosing. When the complexes were administered three times intranasally ( n = 10), no antibodies against the complex were detected in blood. Plasma from mice dosed with saline, nontargeted complex or naked DNA did not react with the ligand, ligand-poly K conjugate or targeted complex. All animals exhibiting human factor IX expression developed antibodies to that transgene by 21 days. Thus, at least three repeat administrations of sec-R-directed molecular conjugates are possible, provided that immune responses to the transgene itself are not limiting.