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.

Strategies for non-viral vectors targeting organs beyond the liver.

In recent years, nanoparticles have evolved to a clinical modality to deliver diverse nucleic acids. Rising interest in nanomedicines comes from proven safety and efficacy profiles established by continuous efforts to optimize physicochemical properties and endosomal escape. However, despite their transformative impact on the pharmaceutical industry, the clinical use of non-viral nucleic acid delivery is limited to hepatic diseases and vaccines due to liver accumulation. Overcoming liver tropism of nanoparticles is vital to meet clinical needs in other organs. Understanding the anatomical structure and physiological features of various organs would help to identify potential strategies for fine-tuning nanoparticle characteristics. In this Review, we discuss the source of liver tropism of non-viral vectors, present a brief overview of biological structure, processes and barriers in select organs, highlight approaches available to reach non-liver targets, and discuss techniques to accelerate the discovery of non-hepatic therapies.

A new era of targeting cystic fibrosis with non-viral delivery of genomic medicines.

Cystic fibrosis (CF) is a complex genetic respiratory disorder that necessitates innovative gene delivery strategies to address the mutations in the gene. This review delves into the promises and challenges of non-viral gene delivery for CF therapy and explores strategies to overcome these hurdles. Several emerging technologies and nucleic acid cargos for CF gene therapy are discussed. Novel formulation approaches including lipid and polymeric nanoparticles promise enhanced delivery through the CF mucus barrier, augmenting the potential of non-viral strategies. Additionally, safety considerations and regulatory perspectives play a crucial role in navigating the path toward clinical translation of gene therapy.

Microfluidic platform enables shear-less aerosolization of lipid nanoparticles for messenger RNA inhalation.

Leveraging the extensive surface area of the lungs for gene therapy, inhalation route offers distinct advantages for delivery. Clinical nebulizers that employ vibrating mesh technology are the standard choice for converting liquid medicines into aerosols. However, they have limitations when it comes to delivering mRNA through inhalation, including severe damage to nanoparticles due to shearing forces. Here, we introduce a novel microfluidic aerosolization platform (MAP) that preserves the structural and physicochemical integrity of lipid nanoparticles, enabling safe and efficient mRNA delivery to the respiratory system. Our results demonstrated the superiority of the novel MAP over the conventional vibrating mesh nebulizer, as it avoided problems such as particle aggregation, loss of mRNA encapsulation, and deformation of nanoparticle morphology. Notably, aerosolized nanoparticles generated by the microfluidic device led to enhanced transfection efficiency across various cell lines. In vivo experiments with mice that inhaled these aerosolized nanoparticles revealed successful, lung-specific mRNA transfection without observable signs of toxicity. This pioneering MAP represents a significant advancement for the pulmonary gene therapy, enabling precise and effective delivery of aerosolized nanoparticles.

Microfluidic Platform Enables Shearless Aerosolization of Lipid Nanoparticles for mRNA Inhalation.

Leveraging the extensive surface area of the lungs for gene therapy, the inhalation route offers distinct advantages for delivery. Clinical nebulizers that employ vibrating mesh technology are the standard choice for converting liquid medicines into aerosols. However, they have limitations when it comes to delivering mRNA through inhalation, including severe damage to nanoparticles due to shearing forces. Here, we introduce a microfluidic aerosolization platform (MAP) that preserves the structural and physicochemical integrity of lipid nanoparticles, enabling safe and efficient delivery of mRNA to the respiratory system. Our results demonstrated the superiority of the MAP over the conventional vibrating mesh nebulizer, as it avoided problems such as particle aggregation, loss of mRNA encapsulation, and deformation of the nanoparticle morphology. Notably, aerosolized nanoparticles generated by the microfluidic device led to enhanced transfection efficiency across various cell lines. In vivo experiments with mice that inhaled these aerosolized nanoparticles revealed successful lung-specific mRNA transfection without observable signs of toxicity. This MAP may represent an advancement for the pulmonary gene therapy, enabling precise and effective delivery of aerosolized nanoparticles.