Hoganson D K, Chandler L A, Fleurbaaij G A, Ying W, Black M E, Doukas J, Pierce G F, Baird A, Sosnowski B A
Selective Genetics, Inc., San Diego, CA 92121, USA.
Hum Gene Ther. 1998 Nov 20;9(17):2565-75. doi: 10.1089/hum.1998.9.17-2565.
Nonviral DNA delivery strategies for gene therapy have generally been limited by a lack of specificity and efficacy. However, ligand-mediated endocytosis can specifically deliver DNA in vitro to cells bearing the appropriate cognate receptors. Similarly, in order to circumvent problems related to efficacy, DNA must encode proteins with high intrinsic activities. We show here that the ligand basic fibroblast growth factor (FGF2) can target FGF receptor-bearing cells with DNA encoding therapeutic proteins. Delivery of genes encoding saporin, a highly potent ribosomal inactivating protein, or the conditionally cytotoxic herpes simplex virus thymidine kinase, a protein that can kill cells by activating the prodrug ganciclovir, is demonstrated. The saporin gene was codon optimized for mammalian expression and demonstrated to express functional protein in a cell-free assay. FGF2-mediated delivery of saporin DNA or thymidine kinase DNA followed by ganciclovir treatment resulted in a 60 and 75% decrease in cell number, respectively. Specificity of gene delivery was demonstrated in competition assays with free FGF2 or with recombinant soluble FGF receptor. Alternatively, when histone H1, a ligand that binds to cell surface heparan sulfate proteoglycans ("low-affinity" FGF receptors), was used to deliver DNA encoding thymidine kinase, no ganciclovir sensitivity was observed. These findings establish the feasibility of using ligands such as FGF2 to specifically deliver genes encoding molecular chemotherapeutic agents to cells.
用于基因治疗的非病毒DNA递送策略通常因缺乏特异性和有效性而受到限制。然而，配体介导的内吞作用可以在体外将DNA特异性地递送至带有相应同源受体的细胞。同样，为了规避与有效性相关的问题，DNA必须编码具有高内在活性的蛋白质。我们在此表明，配体碱性成纤维细胞生长因子（FGF2）可以将携带治疗性蛋白质编码DNA靶向至带有FGF受体的细胞。本文展示了编码皂草素（一种高效核糖体失活蛋白）或条件性细胞毒性单纯疱疹病毒胸苷激酶（一种可通过激活前药更昔洛韦杀死细胞的蛋白质）的基因的递送。皂草素基因经过密码子优化以用于哺乳动物表达，并在无细胞检测中证明可表达功能性蛋白质。FGF2介导的皂草素DNA或胸苷激酶DNA递送，随后进行更昔洛韦处理，分别导致细胞数量减少60%和75%。在与游离FGF2或重组可溶性FGF受体的竞争检测中证明了基因递送的特异性。另外，当使用与细胞表面硫酸乙酰肝素蛋白聚糖结合的配体组蛋白H1（“低亲和力”FGF受体）来递送编码胸苷激酶的DNA时，未观察到更昔洛韦敏感性。这些发现确立了使用FGF2等配体将编码分子化疗药物的基因特异性递送至细胞的可行性。
