RESEARCH ARTICLE Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles A Crespo 1 , A Peydro ´ 2 , F Dası ´ 1 , M Benet 1 , JJ Calvete 3 , F Revert 1 and SF Alin ˜o 1 1 Departamento de Farmacologı ´a, Valencia, Spain; 2 Departamento de Patologı ´a, Facultad de Medicina, Universidad de Valencia, Valencia, Spain; and 3 Instituto de Biomedicina (CSIC), Valencia, Spain The present study contributes to clarify the mechanism underlying the high efficacy of hepatocyte gene transfer mediated by hydrodynamic injection. Gene transfer experi- ments were performed employing the hAAT gene, and the efficacy and differential identification in mouse plasma of human transgene versus mouse gene was assessed by ELISA and proteomic procedures, respectively. By applying different experimental strategies such as cumulative dose– response efficacy, hemodynamic changes reflected by venous pressures, intravital microscopy, and morphological changes established by transmission electron microscopy, we found that: (a) cumulative multiple doses of transgene by hydrodynamic injection are efficient and well tolerated, resulting in therapeutic plasma levels of hAAT; (b) hydro- dynamic injection mediates a transient inversion of intra- hepatic blood flow, with circulatory stasis for a few minutes mainly in pericentral vein sinusoids; (c) transmission electron microscopy shows hydrodynamic injection to promote massive megafluid endocytic vesicles among hepatocytes around the central vein but not in hepatocytes around the periportal vein. We suggest that the mechanism of hydro- dynamic liver gene transfer involves transient inversion of intrahepatic flow, sinusoidal blood stasis, and massive fluid endocytic vesicles in pericentral vein hepatocytes. Gene Therapy (2005) 12, 927–935. doi:10.1038/ sj.gt.3302469; Published online 24 February 2005 Keywords: gene delivery; liver; alpha-1 antitrypsin; genomic DNA; nonviral vector; fluid endocytosis Introduction The liver is a large and extremely complex gland with a great variety of functions, including bile formation, the metabolism of carbohydrate, fat and proteins, and blood filtration. With the exception of gammaglobulins produced by lymphoid tissue plasma cells, all plasma proteins are formed by hepatocytes. Thus, albumin (Alb), alpha-1-antitrypsin, fibrinogen, coagulation factors, and some other proteins are synthesized in the rough endoplasmic reticulum of hepatocytes, transported to the cell surface via the Golgi apparatus, and secreted into the bloodstream. As many disorders of liver metabolism are caused by monogenic defects, the liver should be considered as an important target organ for gene therapy; 1–2 the development of safe and efficient proce- dures for liver gene delivery constitutes a crucial step in this sense. Since naked DNA is the simplest and safest gene delivery system, 3 multiple approaches have been attempted for in vivo liver gene delivery, including intraportal injection, 4 increased DNA retention time in the liver, 5 electrogene transfer, 6 hydrodynamic i.v. injec- tion, 7,8 and mechanical liver massage. 9 However, hydro- dynamic transfection has become widely used 10 because of its simplicity and high efficiency. By employing this procedure, high efficiencies of human alpha-1 antitryp- sin gene transfer have been described, 11,12 and recently we have been able to demonstrate 13 that long-term therapeutic levels of human alpha-1-antitrypsin in mouse plasma can be achieved by this procedure. The important efficacy of nonviral DNA gene delivery mediated by hydrodynamic injection opens new per- spectives for future safe applications in liver gene therapy. Tolerance to cumulative multiple doses is an important advantage for clinical practice, but the efficacy and tolerance to cumulative transgene dose employing the hydrodynamic procedure is poorly known yet. In addition, the procedure has serious limitations for application in clinical practice, and further improvement of the technique requires elucidation of the mechanism underlying the high efficacy of gene transfer – with the aim of combining the mechanistic principle of hydro- dynamic injection with established medical procedures. In this sense, it has recently been suggested that increased pressure in the liver microcirculation produces transient (5–10 min) membrane defects 14 or pores 15 through which naked DNA can penetrate the liver cells through simple diffusion. However, the exact mechanism by which the hepatocyte membrane remains permeable for several minutes, allowing DNA to cross the cell membrane without causing cell death, due to ruptured intracellular ionic homeostasis, remains to be clarified. In the present work, we show good mouse tolerance and efficacy of cumulative hydrodynamic doses of hAAT Received 15 September 2004; accepted 13 December 2004; published online 24 February 2005 Correspondence: Professor SF Alin ˜ o, Departamento de Farmacologı ´a, Facultad de Medicina, Blasco Iba ´n ˜ez 15, 46010 Valencia, Spain Gene Therapy (2005) 12, 927–935 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt