Isolated limb perfusion for local gene delivery: efficient and targeted adenovirus-mediated gene transfer into soft tissue sarcomas

OBJECTIVE: To evaluate the potential of isolated limb perfusion (ILP) for efficient and tumor-specific adenovirus-mediated gene transfer in sarcoma-bearing rats. SUMMARY BACKGROUND DATA: A major concern in adenovirus-mediated gene therapy in cancer is the transfer of genes to organs other than the tumor, especially organs with a rapid cell turnover. Adjustment of the vector delivery route might be an option creating tumor specificity in therapeutic gene expression. METHODS: Rat hind limb sarcomas (5-10 mm) were transfected with recombinant adenoviruses. Intratumoral luciferase expression after ILP was compared with systemic administration, regional infusion, or intratumoral injection using a similar dose of adenoviruses carrying the luciferase marker gene. Localization studies using lacZ as a marker gene were performed to evaluate the intratumoral distribution of transfected cells after both ILP and intratumoral injection. RESULTS: Intratumoral luciferase activity after ILP or intratumoral administration was significantly higher compared with regional infusion or systemic administration. After ILP, luciferase gene expression was minimal in extratumoral organs, whether outside or inside the isolated circuit. Localization studies demonstrated that transfection was confined to tumor cells lying along the needle track after intratumoral injection, whereas after ILP, lacZ expression was found in viable tumor cells and in the tumor-associated vasculature. CONCLUSIONS: Using ILP, efficient and tumor-specific gene transfection can be achieved. The ILP technique might be useful for the delivery of recombinant adenoviruses carrying therapeutic gene constructs to enhance tumor control

Role of the low density lipoprotein receptorrelated protein (lrp) in the clearance and liver uptake of recombinant single chain urokinase-type plasminogen activator (rscu-pa) in rats

Urokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, E.coli produced rscu-PA is recognized by LRP on parenchyma! liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA. A preinjection of the LRP inhibitor GSTRAP reduced the maximal liver uptake of IMl-rscu-PA from 50 to 30 % of the injected dose and decreased the clearance from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10 % of the liver uptake, respectively. The 40 % reduction in liver uptake of rscu-PA by preinjection of GST-RAP was caused by a 91 % and 62 % reduction in the uptake by the parenchymal and Kupffer cells, respectively. Deletion of residue 11 -135 from rscu-PA (=delta125-rscu-PA) resulted in a 80 % reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for the uptake of 60.2, 32.9 and 7.0 % of 125l-delta125-rscu-PA in the liver. Preinjection of GST-RAP completely reduced the liver uptake and reduced the clearance to 0.79 ml/min. Treatment of Kupffer cells with pl-PLC reduced the binding of rscu-PA by 40 %, indicating the involvement of u-PAR in the recognition of rscu-PA by the Kupffer cells. Our results demonstrate that in vivo LRP is responsible for the parenchymal liver cell mediated uptake of rscu-PA and for 60 % of the Kupffer cell interaction. It is also shown that u-PAR is involved in the Kupffer cell recognition of rscu-PA

Characterization of the interaction between urinary urokinase and the asialoglycoprotein receptor of liver cells

Urokinase-type plasminogen activator (u-PA) is rapidly cleared from the circulation with a half-life of a few minutes. We have previously shown that non-glycosylated recombinant u-PA is recognized by LRP, whereas urinary u-PA is recognized by the asialoglycoprotein receptor (ASGPr) on liver parenchymal cells. In the present study we biochemically characterized the latter interaction. The ASGPr was isolated and purified from rat liver tissue and a binding assay was developed. In this assay urinary uPA specifically bound to the receptor with apparent Kd ≈ 30 nM. Specific antibodies against the receptor completely blocked the binding of u-PA. In line with the known carbohydrate specificity of the ASGPr, D-GalNAc proved to be the most effective inhibitor of u-PA binding from a series of monosaccharides, followed by D-Gal and L-Fuc, whereas D-GlcNAc was ineffective. Recent literature showed that the N-linked oligosaccharides of urinary u-PA do not terminate with the common Gal-GlcNAc element but with a GalNAc-GlcNAc element, which is partially sulfated (Eur. J. Biochem. 1995, 228:1009). Sulfated forms of u-PA were separated from non-sulfated forms by using the lectin Wisteria floribunda agglutinin. Only the non-sulfated forms of u-PA (30 % of the total) appeared to bind to the ASGPr. We conclude that a fraction of urinary u-PA bears oligosaccharides which are specifically recognized by the asialoglycoprotein receptor on liver cells