Hydrodynamics-based liver transfection achieves gene silencing of CB1 using short hairpin RNA plasmid in cirrhotic rats Academic Article in Scopus uri icon

abstract

  • © 2020 Díaz-Rivera et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background There is a correlation between the endocannabinoid system and hepatic fibrosis based on the activation of CB1 and CB2 receptors; where CB1 has profibrogenic effects. Gene therapy with a plasmid carrying a shRNA for CB1 delivered by hydrodynamic injection has the advantage of hepatic tropism, avoiding possible undesirable effects of CB1 pharmacological inhibition. Objective To evaluate hydrodynamics-based liver transfection in an experimental model of liver cirrhosis of a plasmid with the sequence of a shRNA for CB1 and its antifibrogenic effects Methods Three shRNA (21pb) were designed for blocking CB1 mRNA at positions 877, 1232 and 1501 (pshCB1-A, B, C). Sequences were cloned in the pENTR¿/U6. Safety was evaluated monitoring CB1 expression in brain tissue. The silencing effect was determined in rat HSC primary culture and CCl4 cirrhosis model. Hydrodynamic injection in cirrhotic liver was through iliac vein and with a dose of 3mg/kg plasmid. Serum levels of liver enzymes, mRNA levels of TGF-ß1, Col IA1 and ¿-SMA and the percentage of fibrotic tissue were analyzed. Results Hydrodynamic injection allows efficient CB1 silencing in cirrhotic livers and pshCB1-B (position 1232) demonstrated the main CB1-silencing. Using this plasmid, mRNA level of fibrogenic molecules and fibrotic tissue considerably decrease in cirrhotic animals. Brain expression of CB1 remained unaltered. Conclusion Hydrodynamics allows a hepatotropic and secure transfection in cirrhotic animals. The sequence of the shCB1-B carried in a plasmid or any other vector has the potential to be used as therapeutic strategy for liver fibrosis.

publication date

  • February 1, 2020