In vivo expression of rat insulin after intravenous administration of the liposome-entrapped gene for rat insulin I.

Abstract

A recombinant plasmid encoding rat preproinsulin I was encapsulated in large liposomes and intravenously injected in rats. Glycemia and blood, splenic, and hepatic insulin were assayed at various times beginning 6 hr after inoculation. The results were compared with controls that had received (i) empty liposomes, (ii) liposomes carrying the Escherichia coli pBR322 plasmid, (iii) the free rat insulin I gene, and (iv) no injection at all. Whereas all controls showed unchanged glucose and insulin levels, the treated animals had, 6 hr after inoculation, a blood glucose level of 72 + 5 mg of glucose/100 ml of blood as compared with 107 + 2 mg/ml for controls. Radioimmunoassay of blood insulin gave 61 + 8 microunits/ml as compared with 43 + 5 microunits/ ml for controls and the spleen and liver values were 242 ± 22 and 204 ± 20 microunits/g of tissue, respectively, as compared with 112+ 20 and 87 + 15 microunits/g in controls. External 7-camera imaging of the organ uptake of "'In-labeled liposomes permitted study ofthe kinetics and extent ofuptake ofthe liposomes by spleen and liver, results that support the findings concerning insulin synthesis in the two organs.Recent studies have shown that, when injected intravenously, liposomes are taken up mainly in the liver and spleen by the macrophages of the reticulo-endothelial system (1-4). Intravenous injection of liposome entrapped. molecules having long latency periods allows the transport of substantial amounts of such molecules to the cellular sites of liposome uptake-i.e., splenic macrophages and.liver Kupffer cells (4, 5).The use ofliposomes to transfer genetic material into culture cells permitted the introduction ofsignificant amounts of exogenous DNA into the cells and led in some cases to expression of the gene thus transported (6-8). We describe in this paper the transport, by liposomes of the gene, encoding rat preproinsulin I to the spleen and liver ofrats. We show its expression in these organs, the release of the, expressed hormone in the blood, and its influence on glycemia. MATERIALS AND METHODSPlasmid Preparations. The recombinant plasmid used in this study, p(gR19,4) contains a 9.4-kilobase EcoRI fragment encoding rat preproinsulin I at the EcoRI site ofpBR322 (9). Plas Tris-HCI, pH 7.4/150 mM NaCl). The two-phase system was sonicated briefly and then the ether was removed at reduced pressure in the rotary evaporator. The liposomes thus formed were treated with DNAse I (Boehringer Mannheim) in the presence of 10 mM of MgCl2 at 37TC (6); the mixture was incubated, the suspension was chromatographed on a Sepharose 4B column, and the 32P radioactivity ofthe DNA was determined. The presence of liposomes was monitored by following the optical density of the samples at 620 nm. The fractions containing the liposomes and the encapsulated DNA were pooled and used for the intravenous inoculations.Entrapment of "'In-Labeled Bleomycin. To determine the location of the large PtdCho/PtdSer/Chol liposomes in the rat organism, liposomes encapsulating "'In...

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