RESEARCH ARTICLE A sensitive noninvasive method for monitoring successful liver-directed gene transfer of the low- density lipoprotein receptor in Watanabe hyperlipidemic rabbits in vivo UJF Tietge 1 , G Cichon 2 , C Bu ¨ ttner 3 , J Genschel 3 , J Heeren 4 , P Gielow 5 , N Grewe 6 , M Dogar 6 , U Beisiegel 4 , MP Manns 6 , H Lochs 2 , W Burchert 7 and HH-J Schmidt 3 1 Department of Medicine and NWFZ, Charite ´ Campus Mitte, Humboldt University, Berlin, Germany; 2 Department of Molecular Cell Biology, Institute for Biology, Humboldt University Berlin at the Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany; 3 Clinic of Gastroenterology, Hepatology, and Endocrinology, Charite ´ Campus Mitte, Humboldt University, Berlin, Germany; 4 Institute of Molecular Cell Biology, Center for Experimental Medicine, University Hospital Eppendorf, Hamburg, Germany; 5 Department of Nuclear Medicine, Hannover Medical School, Hannover, Germany; 6 Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany; and 7 Institute of Molecular Biophysics, Radiopharmacy and Nuclear Medicine, Heart and Diabetes Center North Rhine-Westphalia, Bad Oeynhausen, Germany Noninvasive tools to quantitate transgene expression directly are a prerequisite for clinical gene therapy. We established a method to determine location, magnitude, and duration of low-densitiy lipoprotein (LDL) receptor (LDLR) transgene expression after adenoviral gene transfer into LDLR-deficient Watanabe hypercholesterolemic rabbits by following tissue uptake of intravenously injected 111 In-labeled LDL using a scintillation camera. Liver-specific tracer uptake was calcu- lated by normalizing the counts measured over the liver to counts measured over the heart that represent the circulating blood pool of the tracer (liver/heart (L/H) ratio). Our results indicate that the optimal time point for transgene imaging is 4 h after the tracer injection. Compared with control virus- injected rabbits, animals treated with the LDLR-expressing adenovirus showed seven-fold higher L/H ratios on day 6 after gene transfer, and had still 4.5-fold higher L/H ratios on day 30. This imaging method might be a useful strategy to obtain reliable data on functional transgene expression in clinical gene therapy trials of familial hypercholesterolemia. Gene Therapy (2004) 11, 574–580. doi:10.1038/sj.gt.3302206 Published online 15 January 2004 Keywords: adenovirus; familial hypercholesterolemia; indium; scintillation camera; kinetics Introduction Clinical gene therapy studies require noninvasive tools to evaluate the efficacy of gene transfer. 1,2 Ideally, such an approach should also allow to quantitate functional expression of the therapeutic transgene in vivo repeti- tively. Thereby, the location, magnitude, and duration of the expression of the therapeutic gene could be mon- itored and followed over time. Most of the studies reporting gene transfer imaging so far were performed in the field of cancer gene therapy and did not directly monitor the expression of the gene of interest. 1–3 The activity of enzyme systems such as herpes simplex virus (HSV) thymidine kinase or cytosine deaminase has been assayed by the use of radiolabeled substrates. 1,3 In addition, bicistronic vector systems containing, besides the gene of interest, a reporter construct that would be accessible to radioisotope imaging have been reported. 2,4 Furthermore, gene therapy vectors have been directly coupled with radio- isotopes 5,6 or contrast media. 7 These approaches, how- ever, allow mainly an assessment of tissue sites of the expression and transduction efficacy of the vector itself and not in the first line of the therapeutic gene. Familial hypercholesterolemia (FH) is one of the most common genetic diseases. 8 It has an autosomal dominant genetic trait with an incidence of affected homozygotes of one in one million of the general population. Patients have defects in the low-density lipoprotein (LDL) receptor (LDLR), develop severe hypercholesterolemia, and die because of complications of premature athero- sclerotic cardiovascular disease. 8 Liver is the clinically most important site of the LDLR expression, since liver transplantation is able to cure FH. 9 Owing to these features, FH was among the first diseases for the development of liver-directed clinical gene therapy strategies. Two established animal models are available, LDLR knockout mice 10 and Watanabe hypercholester- olemic (WHHL) rabbits. 11 The latter is more closely resembling human disease, because of the distribution of the cholesterol between lipoprotein subclasses (LDL:HDL ratio) in the rabbits, which is most likely due to the presence of cholesteryl ester transfer protein, and because of their susceptibility to spontaneous Received 17 May 2003; accepted 21 October 2003 Correspondence: Dr med H Schmidt, Clinic of Gastroenterology, Charite ´ Campus Mitte, Schumannstr. 20/21, Berlin D-10117, Germany Gene Therapy (2004) 11, 574–580 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $25.00 www.nature.com/gt