Medical Student Queen's University Queen's University Toronto, Ontario, Canada
Background:
Background: Fontan-associated liver disease (FALD) significantly impacts long-term survival and quality of life in single-ventricle patients who have undergone the Fontan procedure. Currently, no targeted molecular therapies exist for FALD, highlighting a substantial unmet clinical need. Previous work from our lab demonstrated the efficacy of a four-protein (4P) therapeutic combination derived from pediatric cardiac mesenchymal stromal cells (MSCs), capable of reversing pathological fibrosis by modulating the HIPPO signaling pathway and inhibiting nuclear translocation of the profibrotic yes-associated protein (YAP). We hypothesize that early detection of hepatocellular senescence combined with targeted intervention using lipid nanoparticle (LNP) encapsulated mRNA encoding these MSC-derived proteins can effectively preempt fibrosis progression in FALD.
METHODS AND RESULTS:
Methods: mRNA constructs encoding MSC therapeutic proteins (MIP-3β, CNTF, IL26, BMP-4) were produced through in vitro transcription. Validation of mRNA involved transfection of human embryonic kidney (HEK) cells utilizing a lipofectamine transfection reagent. Western blots were completed to assess transfection efficiency and protein expression. We constructed a rat model of FALD through inferior vena cava banding to simulate hepatic venous hypertension, with evaluation of hepatic senescence markers (β-galactosidase staining) and fibrosis development (Masson's Trichrome staining) at multiple time points post-banding.
Results: mRNA expression vectors encoding proteins (MIP-3β, CNTF, IL26, and BMP-4) with C-Terminal tags (HA, MYC, Flag, and V5) were produced for in vitro expression. Preliminary in vitro validation demonstrated expression and secretion of the proteins in HEK cells through western blot analysis. Preliminary in vitro validation demonstrated high transfection efficiency, robust expression of MSC-derived proteins, and appropriate secretory pathway localization. Validation of our rat model pre-transfection showed hepatic senescence markers were consistently detectable at four weeks and eight weeks post-banding, prior to significant fibrosis, establishing a clinically relevant therapeutic intervention window.
Conclusion:
Conclusion: Our preliminary findings support the feasibility of our mRNA constructs to produce the desired protein in vitro. The early detection of hepatocellular senescence as a pre-fibrotic marker represents a critical advancement, providing a novel diagnostic and therapeutic window. Upcoming work will involve validation of mRNA transfection utilizing LNP-mRNA for expression of the proteins using in vitro and in vivo models. This research advances toward establishing the first targeted molecular therapy for FALD, with significant potential to improve patient outcomes and reduce reliance on liver transplantation among congenital heart disease patients.
