Delivering genetic medicines to photoreceptors with lipid nanoparticles

Project Summary Inherited retinal diseases (IRDs) are a heterogeneous group of genetic diseases that lead to loss of vision and often progress to blindness. As a group, IRDs are linked to over 280 genes and affect ~4.5 million people worldwide. In all IRDs, the ultimate cause of vision loss is degeneration of light-sensing photoreceptor (PR) cells and, in most cases, genetic correction is needed specifically in PRs. Genome editing using the CRISPR-Cas9 toolkit has the potential to correct mutations directly in the patient’s DNA. Unfortunately, lack of a safe, efficient delivery vehicle to PRs is a major barrier to developing gene therapies for IRDs. Viral gene therapies for IRDs are in clinical testing but they have limited cargo size and induce a strong immune response. Lipid nanoparticles (LNPs) are the most clinically advanced, non-viral nucleic acid delivery vehicle. LNPs form a “bubble” composed of different lipids (and other compounds) that envelop nucleic acids and provide robust and safe delivery in vivo. To address this PR-specific delivery barrier, EnterX Biosciences is developing novel PR-specific LNPs to deliver RNA-based gene editing components based on our best-in-class, proprietary lipid technology. Recently, we demonstrated the first and only use of lipid-based delivery to transfect PRs with mRNA in vivo, which was accomplished by attaching a small proprietary peptide targeting moiety to the LNP. Our novel, proprietary ionizable lipid, NTRX-7, can also generate LNPs that deliver mRNA to PRs. Combining this novel lipid chemistry and targeting peptide could enable gene editing in PRs. To improve PR-specific delivery of gene editors, we have two major Aims: 1) investigate efficiency of gene editor delivery via LNPs using NTRX lipids and a targeting peptide in PRs; and 2) deploy top LNP candidates to quantify gene editing efficiency and retinal toxicity. These experiments will identify the top LNP candidate(s) capable of safe, efficient delivery of RNA gene editing components for immediate follow up studies of gene editing in specific IRDs. In Aim 1, we will screen LNP delivery of gene editors to PRs. First, in Aim 1A, we will use DNA barcoding to rapidly identify the most potent derivatives of NTRX-7 LNPs and evaluate which LNPs have suitable delivery efficiency to PRs via subretinal injection in mice. The most potent LNP formulations identified by barcoding will then be formulated with Cas9 mRNA and gRNA and injected subretinally. By sectioning and immunostaining Ai9 reporter mouse retinas, we will assay the number of tdTomato+ PRs to determine the spatial extent of gene editing (successful editing will “allow” tdTomato expression in Ai9 mice) and determine the most potent lipids. In Aim 1B, we will similarly test if adding the peptide targeting moiety to the LNP surface can further improve gene editing efficacy and fine-tune PR specificity. Finally, in Aim 2, the top LNP candidates from Aim 1 will be tested longitudinally for retinal toxicity and to quantify PR genome editing in reporter Nrl-GFP mice (here, gene editing will knock out GFP expression). Successfully completing these Aims sets the table for further testing in animal models for retinal diseases and unlocks therapeutic development for a major, unmet health need as well as the clinical market for IRD therapies.