Thiophene-based lipids for mRNA delivery to pulmonary and retinal tissues.

Lipid nanoparticles (LNPs) largely rely on ionizable lipids to yield successful nucleic acid delivery via electrostatic disruption of the endosomal membrane. Here, we report the identification and evaluation of ionizable lipids containing a thiophene moiety (Thio-lipids). The Thio-lipids can be readily synthesized via the Gewald reaction, allowing for modular lipid design with functional constituents at various positions of the thiophene ring. Through the rational design of ionizable lipid structure, we prepared 47 Thio-lipids and identified some structural criteria required in Thio-lipids for efficient mRNA (messenger RNA) encapsulation and delivery in vitro and in vivo. Notably, none of the tested lipids have a pH-response profile like traditional ionizable lipids, potentially due to the electron delocalization in the thiophene core. Placement of the tails and localization of the ionizable headgroup in the thiophene core can endow the nanoparticles with the capability to reach various tissues. Using high-throughput formulation and barcoding techniques, we optimized the formulations to select two top lipids-20b and 29d-and investigated their biodistribution in mice. Lipid 20b enabled LNPs to transfect the liver and spleen, and 29d LNP transfected the lung and spleen. Unexpectedly, LNP with lipid 20b was especially potent in mRNA delivery to the retina with no acute toxicity, leading to the successful delivery to the photoreceptors and retinal pigment epithelium in non-human primates.

Preformed Vesicle Approach to LNP Manufacturing Enhances Retinal mRNA Delivery.

Complete encapsulation of nucleic acids by lipid-based nanoparticles (LNPs) is often thought to be one of the main prerequisites for successful nucleic acid delivery, as the lipid environment protects mRNA from degradation by external nucleases and assists in initiating delivery processes. However, delivery of mRNA via a preformed vesicle approach (PFV-LNPs) defies this precondition. Unlike traditional LNPs, PFV-LNPs are formed via a solvent-free mixing process, leading to a superficial mRNA localization. While demonstrating low encapsulation efficiency in the RiboGreen assay, PFV-LNPs improved delivery of mRNA to the retina by up to 50% compared to the LNP analogs across several benchmark formulations, suggesting the utility of this approach regardless of the lipid composition. Successful mRNA and gene editors' delivery is observed in the retinal pigment epithelium and photoreceptors and validated in mice, non-human primates, and human retinal organoids. Deploying PFV-LNPs in gene editing experiments result in a similar extent of gene editing compared to analogous LNP (up to 3% on genomic level) in the Ai9 reporter mouse model; but, remarkably, retinal tolerability is significantly improved for PFV-LNP treatment. The study findings indicate that the LNP formulation process can greatly influence mRNA transfection and gene editing outcomes, improving LNP treatment safety without sacrificing efficacy.

Development of a translatable gene augmentation therapy for CNGB1-retinitis pigmentosa.

In this study, we investigate a gene augmentation therapy candidate for the treatment of retinitis pigmentosa (RP) due to cyclic nucleotide-gated channel beta 1 (CNGB1) mutations. We use an adeno-associated virus serotype 5 with transgene under control of a novel short human rhodopsin promoter. The promoter/capsid combination drives efficient expression of a reporter gene (AAV5-RHO-eGFP) exclusively in rod photoreceptors in primate, dog, and mouse following subretinal delivery. The therapeutic vector (AAV5-RHO-CNGB1) delivered to the subretinal space of CNGB1 mutant dogs restores rod-mediated retinal function (electroretinographic responses and vision) for at least 12 months post treatment. Immunohistochemistry shows human CNGB1 is expressed in rod photoreceptors in the treated regions as well as restoration of expression and trafficking of the endogenous alpha subunit of the rod CNG channel required for normal channel formation. The treatment reverses abnormal accumulation of the second messenger, cyclic guanosine monophosphate, which occurs in rod photoreceptors of CNGB1 mutant dogs, confirming formation of a functional CNG channel. In vivo imaging shows long-term preservation of retinal structure. In conclusion, this study establishes the long-term efficacy of subretinal delivery of AAV5-RHO-CNGB1 to rescue the disease phenotype in a canine model of CNGB1-RP, confirming its suitability for future clinical development.

Peptide-guided lipid nanoparticles deliver mRNA to the neural retina of rodents and nonhuman primates.

Lipid nanoparticle (LNP)-based mRNA delivery holds promise for the treatment of inherited retinal degenerations. Currently, LNP-mediated mRNA delivery is restricted to the retinal pigment epithelium (RPE) and Müller glia. LNPs must overcome ocular barriers to transfect neuronal cells critical for visual phototransduction, the photoreceptors (PRs). We used a combinatorial M13 bacteriophage-based heptameric peptide phage display library for the mining of peptide ligands that target PRs. We identified the most promising peptide candidates resulting from in vivo biopanning. Dye-conjugated peptides showed rapid localization to the PRs. LNPs decorated with the top-performing peptide ligands delivered mRNA to the PRs, RPE, and Müller glia in mice. This distribution translated to the nonhuman primate eye, wherein robust protein expression was observed in the PRs, Müller glia, and RPE. Overall, we have developed peptide-conjugated LNPs that can enable mRNA delivery to the neural retina, expanding the utility of LNP-mRNA therapies for inherited blindness.

An improved protocol for generation and characterization of human-induced pluripotent stem cell-derived retinal pigment epithelium cells.

We present an optimized protocol for guided differentiation of retinal pigment epithelium (RPE) cells from human-induced pluripotent stem cells (iPSC). De novo-generated RPE cells are mature, polarized, and mimic the cellular and molecular profile of primary RPE; they are also suitable for in vivo cell transplantation studies. The protocol includes an enrichment step, making it useful for large-scale GMP manufacturing. RPE cells produced following this protocol are appropriate for cell replacement therapy for macular degeneration and disease modeling. For complete details on the use and execution of this protocol, please refer to Surendran et al. (2021).