Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles

Abstract

The present study contributes to clarify the mechanism underlying the high efficacy of hepatocyte gene transfer mediated by hydrodynamic injection. Gene transfer experiments 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 doseresponse 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) hydrodynamic injection mediates a transient inversion of intrahepatic 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 hydrodynamic 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.

DNA delivery to ‘ex vivo’ human liver segments

“…2,[8][9][10] However, the systemic perturbation resulting from hydrodynamic injection remains a serious limitation for clinical application, as it can lead to circulatory collapse and even death. To avoid this adverse effect, we have described a retrograde cathetermediated gene delivery procedure 5 with the aim of locally reproducing the intrahepatic conditions mediated by tail vein hydrodynamic injection.…”

“…Also, morphological changes were associated with the intrahepatic pressures, mainly an increased diameter of the sinusoids and Disse space, as well as increased endothelial disruption and the presence of abundant endocytic vesicles in the apical pole of hepatocytes. 8 The rapid increase in both the inferior cava and portal vein pressures mediated by hydrodynamic injection suggests a low vascular resistance, but pressure in the inferior cava vein remains higher for fewer minutes, resulting in an inverted sinusoidal flow. More recently, it has been proposed 17 that during hydrodynamic injection, retrograde DNA solution reaches the portal vein, where the vascular resistance of the intestinal, splenic and pancreatic capillary beds provides efficient outflow obstruction from the portal vein, and thus outflow obstruction is a necessary condition for effective hydrodynamic gene delivery to individual liver segments or lobes.…”

“…The exact mechanism of gene delivery mediated by hydrodynamic injection remains unknown, but two routes of entry of DNA plasmids into hepatocytes have been suggested: plasma membrane rupture 19 and increased membrane permeation, 5,8,20,21 possibly through endocytic vesicles. In the present study we confirm that hydrodynamic injection mediates an increase in sinusoid diameter, with an endothelium that combines both continuous and discontinuous zones, although in no case was generalized disruption observed.…”

“…2,4,5,8 To confirm whether these morphological changes also occur in humans, the liver segments were retrogradely injected at a 20 ml s -1 flow rate with buffered 2.5% glutaraldehyde solution. Then, tissue samples were collected for transmission electron microscopy (TEM).…”

Minimally invasive and selective hydrodynamic gene therapy of liver segments in the pig and human

“…Explanations for the hydrodynamic gene transfer observed have been increase in membrane permeability and endocytic vesicle formation by the hepatocyte. [3][4][5][6] Presently, there is no evidence of toxicity or immune elimination of the transgene product, with in vivo evidence for long-term expression after HGT. 7 The transfer of HGT from the small to large animal model has been problematic with concerns that the massive fluid shifts involved would precipitate heart failure.…”