Resina Sarah, Abes Saïd, Turner John J, Prevot Paul, Travo Adrian, Clair Philippe, Gait Michael J, Thierry Alain R, Lebleu Bernard
Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Département de Défenses Antivirales et Antitumorales, Université Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France.
Int J Pharm. 2007 Nov 1;344(1-2):96-102. doi: 10.1016/j.ijpharm.2007.04.039. Epub 2007 May 17.
Synthetic oligonucleotides offer interesting prospects for the control of gene expression but clinical applications have been severely limited by their poor bioavailability. Cationic lipids have been widely used for the delivery of charged oligonucleotide (ON) analogues but most of the commercial formulations are toxic and poorly stable in the presence of serum proteins. We have developed a DOGS/DOPE liposome formulation named DLS (for delivery liposomal system), that allows for the efficient nuclear delivery of negatively charged antisense ON analogues as monitored by fluorescence microscopy and by their ability to correct deficient pre-mRNA splicing, even in serum-supplemented cell culture. Uncharged DNA mimics such as peptide nucleic acids (PNA), or phosphorodiamidate morpholino (PMO) ON are particularly interesting for their high metabolic stability and affinity for complementary RNA targets but they cannot be delivered with cationic lipids. Cell penetrating peptides (CPP), such as Tat or penetratin, have been used widely as conjugates for the delivery of various biomolecules and might be appropriate for neutral ON analogues. However, entrapment within endocytic vesicles severely limits the efficiency of PNA delivery by CPPs in the absence of endosomolytic drugs, such as chloroquine. The conjugation of new arginine-rich CPPs to PNA allows efficient nuclear delivery in the absence of chloroquine as monitored in a splicing correction assay. Both strategies have their advantages but DLS-mediated delivery remains more efficient than CPP delivery for the nuclear targeting of splice correcting ON analogues in vitro.
合成寡核苷酸为基因表达的控制提供了有趣的前景，但临床应用因其生物利用度差而受到严重限制。阳离子脂质已被广泛用于带电荷的寡核苷酸（ON）类似物的递送，但大多数商业制剂有毒，并且在血清蛋白存在下稳定性较差。我们开发了一种名为DLS（递送脂质体系统）的DOGS/DOPE脂质体制剂，通过荧光显微镜监测以及其纠正缺陷前体mRNA剪接的能力（即使在补充血清的细胞培养中），该制剂能够有效地将带负电荷的反义ON类似物递送至细胞核。不带电荷的DNA模拟物，如肽核酸（PNA）或吗啉代磷酰胺二胺（PMO）ON，因其高代谢稳定性和对互补RNA靶标的亲和力而特别受关注，但它们不能用阳离子脂质递送。细胞穿透肽（CPP），如Tat或穿膜肽，已被广泛用作各种生物分子递送的缀合物，可能适用于中性ON类似物。然而，在没有溶酶体药物（如氯喹）的情况下，内吞囊泡内的包裹严重限制了CPP对PNA的递送效率。在剪接校正试验中监测到，将新的富含精氨酸的CPP与PNA缀合可在没有氯喹的情况下实现高效的细胞核递送。两种策略都有其优点，但在体外对剪接校正ON类似物进行细胞核靶向时，DLS介导的递送仍然比CPP递送更有效。
