Lipid nanoparticles for delivery of messenger RNA to the back of the eye

Abstract

Retinal gene therapy has had unprecedented success in generating treatments that can halt vision loss. However, immunogenic response and long-term toxicity with the use of viral vectors remain a concern. Non-viral vectors are relatively non-immunogenic, scalable platforms that have limited success with DNA delivery to the eye. Messenger RNA (mRNA) therapeutics has expanded the ability to achieve high gene expression while eliminating unintended genomic integration or the need to cross the restrictive nuclear barrier. Lipid-based nanoparticles (LNPs) remain at the forefront of potent delivery vectors for nucleic acids. Herein, we tested eleven different LNP variants for their ability to deliver mRNA to the back of the eye. LNPs that contained ionizable lipids with low pKa and unsaturated hydrocarbon chains showed the highest amount of a reporter gene transfection in the retina. The kinetics of gene expression showed a rapid onset (within 4 h) that persisted for 96 h. The gene delivery was cell-type specific with majority of the expression in the retinal pigmented epithelium (RPE) and limited expression in the Müller glia. LNP-delivered mRNA can be used to treat monogenic retinal degenerative disorders of the RPE. The transient nature of mRNA-based therapeutics makes it desirable for applications that are directed towards retinal reprogramming or genome editing. Overall, non-viral delivery of RNA therapeutics to diverse cell types within the retina can provide transformative new approaches to prevent blindness.

The effects of PEGylation on LNP based mRNA delivery to the eye

“…To optimize the existing FDA approved LNPs for the retina, studies are needed that investigate pharmacokinetics and intracellular mechanisms of LNPs post-ocular delivery. Our studies, first evaluating many cationic and ionizable lipids [ 13 ], and now investigating the impacts of size and PEGylation, aim to understand the critical LNP components needed for optimal retina internalization and penetration to the eye. In this paper, we show that particles with less PEG (0.5%) and larger in size (~150 nm) facilitate the highest levels of protein expression post-subretinal and intravitreal delivery.…”

“…Prior to injections, mice were topically administered 0.5% proparacaine, 1% tropicamide, and 2.5% phenylephrine and anesthetized with ketamine (100 mg/kg)/xylazine (10 mg/kg). Our subretinal injection procedure has been previously described elsewhere [ 13 ]. For intravitreal injections, 2.5% hypromellose was placed over the eye and a 30-gauge needle was used to make an incision in the limbus.…”

“…Mice were injected intraperitoneally with 150 mg of Luciferin/kg body weight according to manufacturer’s protocol [ 13 ]. Bioluminescent imaging was conducted on the IVIS Spectrum In Vivo Imaging System (PerkinElmer).…”

“…LNPs are comprised of structural lipids, cationic or ionizable lipids, PEG-lipids and cholesterol (50:10:1.5:38.5% mol ratios), all which can be rationally designed for optimal cell-specific gene delivery. Previously, we evaluated 11 different cationic or ionizable lipids for their ability to transfect the retina post-subretinal delivery [ 13 ]. We found that LNPs containing the ionizable lipid, MC3 (pKa = 6.5), most efficiently transfect the retinal pigment epithelium (RPE).…”

Nanomedicines to Deliver mRNA: State of the Art and Future Perspectives

“…Protein replacement therapies based on IVT mRNA have been mainly directed to the liver [201], lungs [202], and heart [203] because of the accessibility of these organs. However, other organs and tissues, such as skin [204], back of the eye [205] or nasal cavity [131] have also been evaluated as target sites. The main applications of this therapy are genetic and rare diseases.…”