Summary

Gaurav Sahay’s lab at Oregon Health and Science University have just published a paper describing a systematic study of lipid nanoparticle (LNP) formulations for delivering mRNA to the retina. Their research has potential implications towards developing gene therapies or gene editing treatments for diseases of the eye such as age-related macular degeneration, inherited retinal degeneration, and glaucoma. LNPs offer a potentially safer approach to delivering CRIPSR components than viral systems that are generally being explored for these applications.

The Sahay lab tested 11 cationic lipids (CLs) with helper lipids, cholesterol, and a PEG-lipid at a fixed molar ratio. They categorized these CLs in terms of their hydrocarbon saturation and amine structure. After formulating these lipids with mRNA using NanoAssemblr Technology they found CLs without double-bonds in the hydrocarbon tails, failed to form stable LNPs. The remaining 7 formulations which contained CLs bearing at least one unsaturated C-C bond in each tail and either a tertiary amine or quaternary amine in the headgroup (labeled as “Group I” and “Group II”, respectively) were selected for testing in mice to determine which is effective in inducing expression of a reporter gene.

They injected the formulations subretinally and they found both Group I and Group II lipids mediated transgene expression in the eye, but found forulations with Group I CLs had protein expression localized to the retina, with a majority in retinal pigment epithelium. Formulations with Group II lipids did not show localization. Overall, higher levels of transgene expression were observed for the Group I lipids. To explain this, the authors point to differences in structure of the resulting LNPs. Group I lipids include MC3, KC2, and DODMA, which are ionizable, while Group II lipids (DOTMA, DOTAP, etc) are permanently cationic and tend to form lamellar structures. Lamellar structures are believed to be less efficient in releasing the payload into the cytoplasm. Additionally, MC3 and KC2 have two double bonds in each hydrocarbon tail which is believed to improve endosomal escape of mRNA compared to singly unsaturated tails as in DODMA, and the reported expression levels are consistent with this understanding.

Subretinal injections cause a bleb to form under the retina, which in 60% of cases resulted in retinal detachment. Hence, the authors intend to develop LNP formulations for intravitreal delivery. This will make such treatments more practical and enable treatment of a wide variety of eye diseases with a genetic cause.

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.