Dataset Information

Hydrodynamics-based liver transfection achieves gene silencing of CB1 using short hairpin RNA plasmid in cirrhotic rats.

ABSTRACT: 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-?1, 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.

SUBMITTER: Diaz-Rivera A

PROVIDER: S-EPMC7018086 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Hydrodynamics-based liver transfection achieves gene silencing of CB1 using short hairpin RNA plasmid in cirrhotic rats.

Díaz-Rivera Adriana A Meza-Ríos Alejandra A Chagoya de Sánchez Victoria V Velasco-Loyden Gabriela G García-Benavides Leonel L Jave-Suarez Luis F LF Monroy-Ramirez Hugo Christian HC Santos-García Arturo A Armendáriz-Borunda Juan J Sandoval-Rodríguez Ana A

PloS one 20200213 2

<h4>Background</h4>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.<h4>Objective</h4>To evaluate hydrodynamics-based liver transfection in an experimental model of liver cirrhosis of ...[more]

PMID: 32053633

Similar Datasets

Project description:In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×10(5)±3.3×10(5) (MC-Luc) versus 0.4×10(5)±0.3×10(5) (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31(+) expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.

Dataset Information

Hydrodynamics-based liver transfection achieves gene silencing of CB1 using short hairpin RNA plasmid in cirrhotic rats.

Publications

Hydrodynamics-based liver transfection achieves gene silencing of CB1 using short hairpin RNA plasmid in cirrhotic rats.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets