Translational Advances of Hydrofection by Hydrodynamic Injection

Abstract

Hydrodynamic gene delivery has proven to be a safe and efficient procedure for gene transfer, able to mediate, in murine model, therapeutic levels of proteins encoded by the transfected gene. In different disease models and targeting distinct organs, it has been demonstrated to revert the pathologic symptoms and signs. The therapeutic potential of hydrofection led different groups to work on the clinical translation of the procedure. In order to prevent the hemodynamic side effects derived from the rapid injection of a large volume, the conditions had to be moderated to make them compatible with its use in mid-size animal models such as rat, hamster and rabbit and large animals as dog, pig and primates. Despite the different approaches performed to adapt the conditions of gene delivery, the results obtained in any of these mid-size and large animals have been poorer than those obtained in murine model. Among these different strategies to reduce the volume employed, the most effective one has been to exclude the vasculature of the target organ and inject the solution directly. This procedure has permitted, by catheterization and surgical procedures in large animals, achieving protein expression levels in tissue close to those achieved in gold standard models. These promising results and the possibility of employing these strategies to transfer gene constructs able to edit genes, such as CRISPR, have renewed the clinical interest of this procedure of gene transfer. In order to translate the hydrodynamic gene delivery to human use, it is demanding the standardization of the procedure conditions and the molecular parameters of evaluation in order to be able to compare the results and establish a homogeneous manner of expressing the data obtained, as 'classic' drugs.

Keywords: gene therapy; hydrodynamic; non-viral; translational.

Figure 1

Hydrodynamic method. The figure shows the mouse normal blood flow and the effects mediated by hydrodynamic injection (indicated by a gap line within tail vein and from inferior vena cava to the liver through hepatic vein). Blood flows from tail vein to heart, who drives it to lungs to be oxygenated, returned to heart and distributed to the entire whole through aortic artery. The liver receives a profuse blood supply from the hepatic artery and portal vein. Blood flow from portal vein to inferior cava vein must cross the liver parenchyma through the hepatic sinusoids. When hydrodynamic injection is performed through tail vein, this large volume (2 mL) drains into inferior cava vein, it results in increased venous pressure that mediates retrograde blood flow into liver sinusoids (arrow with dashed line). This permits the gene accessing the liver. Employing different experimental strategies, the hydrofection mechanism has been suggested to involve transient inversion of intrahepatic blood flow and massive fluid endocytic vesicles in hepatocytes, mainly in those distributed around the central vein. The volume stays immobilized but pulsatile until heart, thanks to heart rate increasing, pumps this volume to bloodstream.

Figure 2

Hydrofection mechanism. The figure shows the mechanism underlying the hydrofection gene transfer to the cells in liver. Upper panel shows a liver lobe unit (right) with its vascular system, which has been enlarged (left) to show the sinusoid vessel detail. Squared area is augmented in lower panel, where the sinusoid vessel organization is showed in detail just after hydrofection. It can be observed that retrovenous injection mediates hydrodynamic force that widens the vessel and virtual Disse space, separates endothelial cells and induces large number of endocytic vesicles on hepatocytes without obvious plasma membrane rupture It suggests that hydrodynamic force mediates DNA delivery to hepatocyte via diffusion process, involving a microfluid uptake process and/or penetration through facilitated permeable sites in the cell membrane. PV: portal vein; CV: Cava vein; HA: hepatic artery.

Figure 3

Catheterization strategies for minimally invasive liver hydrofection ‘in vivo.’ Schematic representation of liver venous vasculature and catheters position. Continuous line represents extrahepatic vessels. Gapped lines represent intrahepatic vasculature. Grey arrows indicate the normal blood flow sense. Black thick arrows indicate the sense of hydrodynamic injection.