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.

MicroRNA137-loaded lipid nanoparticles regulate synaptic proteins in the prefrontal cortex.

Genome-wide association studies indicate that allele variants in MIR137, the host gene of microRNA137 (miR137), confer an increased risk of schizophrenia (SCZ). Aberrant expression of miR137 and its targets, many of which regulate synaptic functioning, are also associated with an increased risk of SCZ. Thus, miR137 represents an attractive target aimed at correcting the molecular basis for synaptic dysfunction in individuals with high genetic risk for SCZ. Advancements in nanotechnology utilize lipid nanoparticles (LNPs) to transport and deliver therapeutic RNA. However, there remains a gap in using LNPs to regulate gene and protein expression in the brain. To study the delivery of nucleic acids by LNPs to the brain, we found that LNPs released miR137 cargo and inhibited target transcripts of interest in neuroblastoma cells. Biodistribution of LNPs loaded with firefly luciferase mRNA remained localized to the mouse prefrontal cortex (PFC) injection site without circulating to off-target organs. LNPs encapsulating Cre mRNA preferentially co-expressed in neuronal over microglial or astrocytic cells. Using quantitative proteomics, we found miR137 modulated glutamatergic synaptic protein networks that are commonly dysregulated in SCZ. These studies support engineering the next generation of brain-specific LNPs to deliver RNA therapeutics and improve symptoms of central nervous system disorders.

Chemistry of Lipid Nanoparticles for RNA Delivery.

Lipid nanoparticles (LNPs) are a type of lipid vesicles that possess a homogeneous lipid core. These vesicles are widely used in small-molecule drug and nucleic acid delivery and recently gained much attention because of their remarkable success as a delivery platform for COVID-19 mRNA vaccines. Nonetheless, the utility of transient protein expression induced by mRNA extends far beyond vaccines against infectious diseases─they also hold promise as cancer vaccines, protein replacement therapies, and gene editing components for rare genetic diseases. However, naked mRNA is inherently unstable and prone to rapid degradation by nucleases and self-hydrolysis. Encapsulation of mRNA within LNPs protects mRNA from extracellular ribonucleases and assists with intracellular mRNA delivery.In this Account, we discuss the core features of LNPs for RNA delivery. We focus our attention on LNPs designed to deliver mRNA; however, we also include examples of siRNA-LNP delivery where appropriate to highlight the commonalities and the dissimilarities due to the nucleic acid structure. First, we introduce the concept of LNPs, the advantages and disadvantages of utilizing nucleic acids as therapeutic agents, and the general reasoning behind the molecular makeup of LNPs. We also briefly highlight the most recent clinical successes of LNP-based nucleic acid therapies. Second, we describe the theory and methods of LNP self-assembly. The common idea behind all of the preparation methods is inducing electrostatic interactions between the nucleic acid and charged lipids and promoting nanoparticle growth via hydrophobic interactions. Third, we break down the LNP composition with special attention to the fundamental properties and purposes of each component. This includes the identified molecular design criteria, commercial sourcing, impact on intracellular trafficking, and contribution to the properties of LNPs. One of the key components of LNPs is ionizable lipids, which initiate electrostatic binding with endosomal membranes and facilitate cytosolic release; however, the roles of other lipid components should not be disregarded, as they are associated with stability, clearance, and distribution of LNPs. Fourth, we review the attributes of LNP constructs as a whole that can heavily influence RNA delivery. These attributes are LNP size, charge, internal structure, lipid packing, lipid membrane hydration, stability, and affinity toward biomacromolecules. We also discuss the specific techniques used to examine these attributes and how they can be adjusted. Finally, we offer our perspective on the future of RNA therapies and some questions that remain in the realm of LNP formulation and optimization.

Nanoparticle-Based Follistatin Messenger RNA Therapy for Reprogramming Metastatic Ovarian Cancer and Ameliorating Cancer-Associated Cachexia.

This study presents the first messenger RNA (mRNA) therapy for metastatic ovarian cancer and cachexia-induced muscle wasting based on lipid nanoparticles that deliver follistatin (FST) mRNA predominantly to cancer clusters following intraperitoneal administration. The secreted FST protein, endogenously synthesized from delivered mRNA, efficiently reduces elevated activin A levels associated with aggressive ovarian cancer and associated cachexia. By altering the cancer cell phenotype, mRNA treatment prevents malignant ascites, delays cancer progression, induces the formation of solid tumors, and preserves muscle mass in cancer-bearing mice by inhibiting negative regulators of muscle mass. Finally, mRNA therapy provides synergistic effects in combination with cisplatin, increasing the survival of mice and counteracting muscle atrophy induced by chemotherapy and cancer-associated cachexia. The treated mice develop few nonadherent tumors that are easily resected from the peritoneum. Clinically, this nanomedicine-based mRNA therapy can facilitate complete cytoreduction, target resistance, improve resilience during aggressive chemotherapy, and improve survival in advanced ovarian cancer.

Leveraging Biological Buffers for Efficient Messenger RNA Delivery via Lipid Nanoparticles.

Lipid nanoparticles containing messenger RNA (mRNA-LNPs) have launched to the forefront of nonviral delivery systems with their realized potential during the COVID-19 pandemic. Here, we investigate the impact of commonly used biological buffers on the performance and durability of mRNA-LNPs. We tested the compatibility of three common buffers─HEPES, Tris, and phosphate-buffered saline─with a DLin-MC3-DMA mRNA-LNP formulation before and after a single controlled freeze-thaw cycle. We hypothesized that buffer composition would affect lipid-aqueous phase separation. Indeed, the buffers imposed structural changes in LNP morphology as indicated by electron microscopy, differential scanning calorimetry, and membrane fluidity assays. We employed in vitro and in vivo models to measure mRNA transfection and found that Tris or HEPES-buffered LNPs yielded better cryoprotection and transfection efficiency compared to PBS. Understanding the effects of various buffers on LNP morphology and efficacy provides valuable insights into maintaining the stability of LNPs after long-term storage.

In vitro and ex vivo strategies for intracellular delivery.

Intracellular delivery of materials has become a critical component of genome-editing approaches, ex vivo cell-based therapies, and a diversity of fundamental research applications. Limitations of current technologies motivate development of next-generation systems that can deliver a broad variety of cargo to diverse cell types. Here we review in vitro and ex vivo intracellular delivery approaches with a focus on mechanisms, challenges and opportunities. In particular, we emphasize membrane-disruption-based delivery methods and the transformative role of nanotechnology, microfluidics and laboratory-on-chip technology in advancing the field.

Matrix stiffness regulates lipid nanoparticle-mRNA delivery in cell-laden hydrogels.

mRNA therapeutics have increased in popularity, largely due to the transient and fast nature of protein expression and the low risk of off-target effects. This has increased drastically with the remarkable success of mRNA-based vaccines for COVID-19. Despite advances in lipid nanoparticle (LNP)-based delivery, the mechanisms that regulate efficient endocytic trafficking and translation of mRNA remain poorly understood. Although it is widely acknowledged that the extracellular matrix (ECM) regulates uptake and expression of exogenous nano-complexed genetic material, its specific effects on mRNA delivery and expression have not yet been examined. Here, we demonstrate a critical role for matrix stiffness in modulating both mRNA transfection and expression and uncover distinct mechano-regulatory mechanisms for endocytosis of mRNA through RhoA mediated mTOR signaling and cytoskeletal dynamics. Our findings have implications for effective delivery of therapeutic mRNA to targeted tissues that may be differentially affected by tissue and matrix stiffness.

Deconvoluting Lipid Nanoparticle Structure for Messenger RNA Delivery.

Lipid nanoparticle (LNP) packaged mRNA vaccines have been deployed against infectious diseases such as COVID-19, yet their structural features remain unclear. Cholesterol, a major constituent within LNPs, contributes to their morphology that influences gene delivery. Herein, we examine the structure of LNPs containing cholesterol derivatives using electron microscopy, differential scanning calorimetry, and membrane fluidity assays. LNPs formulated with C24 alkyl derivatives of cholesterol show a polymorphic shape and various degrees of multilamellarity and lipid partitioning, likely due to phase separation. The addition of methyl and ethyl groups to the C24 alkyl tail of the cholesterol backbone induces multilamellarity (>50% increase compared to cholesterol), while the addition of a double bond induces lipid partitioning (>90% increase compared to cholesterol). LNPs with multilamellar and faceted structures, as well as a lamellar lipid phase, showed higher gene transfection. Unraveling the structure of mRNA-LNPs can enable their rational design toward enhanced gene delivery.

Lipid Nanoparticle-Delivered Chemically Modified mRNA Restores Chloride Secretion in Cystic Fibrosis.

The promise of gene therapy for the treatment of cystic fibrosis has yet to be fully clinically realized despite years of effort toward correcting the underlying genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR). mRNA therapy via nanoparticle delivery represents a powerful technology for the transfer of genetic material to cells with large, widespread populations, such as airway epithelia. We deployed a clinically relevant lipid-based nanoparticle (LNP) for packaging and delivery of large chemically modified CFTR mRNA (cmCFTR) to patient-derived bronchial epithelial cells, resulting in an increase in membrane-localized CFTR and rescue of its primary function as a chloride channel. Furthermore, nasal application of LNP-cmCFTR restored CFTR-mediated chloride secretion to conductive airway epithelia in CFTR knockout mice for at least 14 days. On day 3 post-transfection, CFTR activity peaked, recovering up to 55% of the net chloride efflux characteristic of healthy mice. This magnitude of response is superior to liposomal CFTR DNA delivery and is comparable with outcomes observed in the currently approved drug ivacaftor. LNP-cmRNA-based systems represent a powerful platform technology for correction of cystic fibrosis and other monogenic disorders.

Boosting Intracellular Delivery of Lipid Nanoparticle-Encapsulated mRNA.

Intracellular delivery of mRNA holds great potential for vaccine1-3 and therapeutic4 discovery and development. Despite increasing recognition of the utility of lipid-based nanoparticles (LNPs) for intracellular delivery of mRNA, particle engineering is hindered by insufficient understanding of endosomal escape, which is believed to be a main limiter of cytosolic availability and activity of the nucleic acid inside the cell. Using a series of CRISPR-based genetic perturbations of the lysosomal pathway, we have identified that late endosome/lysosome (LE/Ly) formation is essential for functional delivery of exogenously presented mRNA. Lysosomes provide a spatiotemporal hub to orchestrate mTOR signaling and are known to control cell proliferation, nutrient sensing, ribosomal biogenesis, and mRNA translation. Through modulation of the mTOR pathway we were able to enhance or inhibit LNP-mediated mRNA delivery. To further boost intracellular delivery of mRNA, we screened 212 bioactive lipid-like molecules that are either enriched in vesicular compartments or modulate cell signaling. Surprisingly, we have discovered that leukotriene-antagonists, clinically approved for treatment of asthma and other lung diseases, enhance intracellular mRNA delivery in vitro (over 3-fold, p < 0.005) and in vivo (over 2-fold, p < 0.005). Understanding LNP-mediated intracellular delivery will inspire the next generation of RNA therapeutics that have high potency and limited toxicity.

Small RNA combination therapy for lung cancer.

MicroRNAs (miRNAs) and siRNAs have enormous potential as cancer therapeutics, but their effective delivery to most solid tumors has been difficult. Here, we show that a new lung-targeting nanoparticle is capable of delivering miRNA mimics and siRNAs to lung adenocarcinoma cells in vitro and to tumors in a genetically engineered mouse model of lung cancer based on activation of oncogenic Kirsten rat sarcoma viral oncogene homolog (Kras) and loss of p53 function. Therapeutic delivery of miR-34a, a p53-regulated tumor suppressor miRNA, restored miR-34a levels in lung tumors, specifically down-regulated miR-34a target genes, and slowed tumor growth. The delivery of siRNAs targeting Kras reduced Kras gene expression and MAPK signaling, increased apoptosis, and inhibited tumor growth. The combination of miR-34a and siRNA targeting Kras improved therapeutic responses over those observed with either small RNA alone, leading to tumor regression. Furthermore, nanoparticle-mediated small RNA delivery plus conventional, cisplatin-based chemotherapy prolonged survival in this model compared with chemotherapy alone. These findings demonstrate that RNA combination therapy is possible in an autochthonous model of lung cancer and provide preclinical support for the use of small RNA therapies in patients who have cancer.

Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates.

siRNA therapeutics have promise for the treatment of a wide range of genetic disorders. Motivated by lipoproteins, we report lipopeptide nanoparticles as potent and selective siRNA carriers with a wide therapeutic index. Lead material cKK-E12 showed potent silencing effects in mice (ED50 ∼ 0.002 mg/kg), rats (ED50 < 0.01 mg/kg), and nonhuman primates (over 95% silencing at 0.3 mg/kg). Apolipoprotein E plays a significant role in the potency of cKK-E12 both in vitro and in vivo. cKK-E12 was highly selective toward liver parenchymal cell in vivo, with orders of magnitude lower doses needed to silence in hepatocytes compared with endothelial cells and immune cells in different organs. Toxicity studies showed that cKK-E12 was well tolerated in rats at a dose of 1 mg/kg (over 100-fold higher than the ED50). To our knowledge, this is the most efficacious and selective nonviral siRNA delivery system for gene silencing in hepatocytes reported to date.

Multiparametric approach for the evaluation of lipid nanoparticles for siRNA delivery.

Nanoparticle-mediated siRNA delivery is a complex process that requires transport across numerous extracellular and intracellular barriers. As such, the development of nanoparticles for efficient delivery would benefit from an understanding of how parameters associated with these barriers relate to the physicochemical properties of nanoparticles. Here, we use a multiparametric approach for the evaluation of lipid nanoparticles (LNPs) to identify relationships between structure, biological function, and biological activity. Our results indicate that evaluation of multiple parameters associated with barriers to delivery such as siRNA entrapment, pKa, LNP stability, and cell uptake as a collective may serve as a useful prescreening tool for the advancement of LNPs in vivo. This multiparametric approach complements the use of in vitro efficacy results alone for prescreening and improves in vitro-in vivo translation by minimizing false negatives. For the LNPs used in this work, the evaluation of multiple parameters enabled the identification of LNP pKa as one of the key determinants of LNP function and activity both in vitro and in vivo. It is anticipated that this type of analysis can aid in the identification of meaningful structure-function-activity relationships, improve the in vitro screening process of nanoparticles before in vivo use, and facilitate the future design of potent nanocarriers.

Combinatorial synthesis of chemically diverse core-shell nanoparticles for intracellular delivery.

Analogous to an assembly line, we employed a modular design for the high-throughput study of 1,536 structurally distinct nanoparticles with cationic cores and variable shells. This enabled elucidation of complexation, internalization, and delivery trends that could only be learned through evaluation of a large library. Using robotic automation, epoxide-functionalized block polymers were combinatorially cross-linked with a diverse library of amines, followed by measurement of molecular weight, diameter, RNA complexation, cellular internalization, and in vitro siRNA and pDNA delivery. Analysis revealed structure-function relationships and beneficial design guidelines, including a higher reactive block weight fraction, stoichiometric equivalence between epoxides and amines, and thin hydrophilic shells. Cross-linkers optimally possessed tertiary dimethylamine or piperazine groups and potential buffering capacity. Covalent cholesterol attachment allowed for transfection in vivo to liver hepatocytes in mice. The ability to tune the chemical nature of the core and shell may afford utility of these materials in additional applications.

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.

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.

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.

Lipid Nanoparticle-Enabled Intracellular Delivery of Prime Editors.

Prime editing is an advanced gene editing platform with potential to correct almost any disease-causing mutation. As genome editors have evolved, their size and complexity have increased, hindering delivery technologies with low-carrying capacity and endosomal escape. We formulated an array of lipid nanoparticles (LNPs) containing prime editors (PEs). We were able to encapsulate PEs in LNPs and confirmed the presence of PE mRNA and two different guide RNAs using HPLC. In addition, we developed a novel reporter cell line for rapid identification of LNPs suited for prime editing. A 54% prime editing rate was observed with enhanced LNPs (eLNPs) containing the cholesterol analog ß-sitosterol at optimal ratios of RNA cargoes. eLNPs displayed a polyhedral morphology and a more fluid membrane state that led to improved endosomal escape, eventually causing onset of editing within 9 h and reaching maximum efficiency after 24 h. Hence, PEs delivered using LNPs can propel a new wave of therapies for many additional targets potentially enabling a range of new applications.