Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery

Abstract

The primary cause of failure for minimally invasive glaucoma surgery (MIGS) is fibrosis in the trabecular meshwork (TM) that regulates the outflow of aqueous humour, and no anti-fibrotic drug is available for intraocular use in MIGS. The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway is a promising anti-fibrotic target. This study aims to utilise a novel lipid nanoparticle (LNP) to deliver MRTF-B siRNA into human TM cells and to compare its effects with those observed in human conjunctival fibroblasts (FF). Two LNP formulations were prepared with and without the targeting peptide cΥ, and with an siRNA concentration of 50 nM. We examined the biophysical properties and encapsulation efficiencies of the LNPs, and evaluated the effects of MRTF-B silencing on cell viability, key fibrotic genes expression and cell contractility. Both LNP formulations efficiently silenced MRTF-B gene and were non-cytotoxic in TM and FF cells. The presence of cΥ made the LNPs smaller and more cationic, but had no significant effect on encapsulation efficiency. Both TM and FF cells also showed significantly reduced contractibility after transfection with MRTF-B siRNA LNPs. In TM cells, LNPs with cΥ achieved a greater decrease in contractility compared to LNPs without cΥ. In conclusion, we demonstrate that the novel CL4H6-LNPs are able to safely and effectively deliver MRTF-B siRNA into human TM cells. LNPs can serve as a promising non-viral gene therapy to prevent fibrosis in MIGS.

The Effect of Cryoprotectants and Storage Conditions on the Transfection Efficiency, Stability, and Safety of Lipid‐Based Nanoparticles for mRNA and DNA Delivery

“…To that end, we synthesized and tested 2 distinct types of lipid‐based nanocarriers; Receptor‐targeted nanocomplexes (RTNs), which comprised of liposome, targeting peptide and nucleic acid, and LNPs utilizing a pH‐sensitive lipid to encapsulate nucleic acids. Both types of NPs have previously demonstrated exceptional transfection efficiencies and low cytotoxicity, by our group and others, [ 41–53 ] thus presenting ideal models for our current and future research. Additionally, a peptide targeting the low‐density lipoprotein receptor (LDLR), peptide AT‐19, was used for the formulation of the LNPs, as it has been previously suggested that the addition of targeting peptides can increase the encapsulation efficiency and overall stability of the LNPs.…”

Glaucoma: Novel antifibrotic therapeutics for the trabecular meshwork

“…The SRF/MRTF pathway is a major cytoskeletal regulator and represents a promising anti-fibrotic target. By utilising novel lipid nanoparticles (LNP), Luo et al (2022) synthesized PEGylated CL4H6-MRTF-B siRNA-loaded LNPs, which can protect siRNA from rapid degradation, with the aim of preventing TM fibrosis after MIGS. The results suggested that the novel CL4H6-LNPs can effectively and safely deliver MRTF-B siRNA into hTM cells, and can significantly reduce the contractibility of TM cells after transfection.…”

“…1 B) ( Dhingra and Bhartiya, 2020 ). However, fibrosis in the TM tissue can adversely affect the implanted stents and is the main cause of failure in MIGS ( Luo et al, 2022 ). Anti-fibrotic therapeutics targeting the TM thus represent a promising strategy in glaucoma management by reducing IOP as well as improving the surgical success rates of MIGS devices.…”

“… 1 μM porcine ( Aebersold and Song, 2022 ) Doubling aqueous humor outflow compared with the vehicle in porcine anterior segment-perfused organ culture model. Nanomedicine Agent Nanomaterial Cell line/tissue Efficiency and Potency against fibrosis siRNA-MRTF-B CL4H6- LNP human TM cell ( Luo et al, 2022 ) Protecting siRNA from rapid degradation, effectively delivering MRTF-B siRNA into human TM cells with no toxicity and significantly reducing contractibility of TM cells. siRNA-CTGF hyaluronan porcine ( Dillinger et al, 2018 ) human ( Dillinger et al, 2018 ) Deeply penetrating into the outflow region of murine by targeting CD44 and can decreasing CTGF-mediated fibrosis by reduction of CTGF to about 50% in the donor cells with high basal CTGF expression.…”

„Minimally invasive glaucoma surgery“ – Kammerwinkelbasierte Verfahren im Vergleich

“…Die vorliegenden Ergebnisse stammen aus den Zulassungsstudien und zeigen eine durchschnittliche Senkung des Augeninnendruckes von 39 % über 2 Jahre bei einer gleichzeitigen Medikamentenreduktion von 2,4 auf 1,4 nach 24 Monaten [66, Zusammenfassend muss jedoch festgestellt werden, dass auch nach mehr als einem Jahrzehnt der MIGS die Evidenz hinsichtlich der Senkung des Augeninnendruckes und/oder der Reduktion der Medikamentenlast der Patienten bei vielen Verfahren noch gering ist und es daher weiterer qualitativ hochwertiger Studien bedarf, um klare Richtlinien für den Einsatz der einzelnen Verfahren abzuleiten. Es wird auch zu klären sein, ob durch eine gezielte Fibrosehemmung im Trabekelmaschenwerk [69] oder im suprachoroidalen Raum [70] die Langzeiterfolge nach unterschiedlichen MIGS-Verfahren verbessert werden können.…”