Transfection Potency of Lipid Nanoparticles Containing mRNA Depends on Relative Loading Levels.

When formulating mRNA into lipid nanoparticles (LNP), various copy numbers of mRNA are encapsulated, leading to a distribution of mRNA loading levels within the LNPs. It is unclear whether the mRNA loading level affects the functional delivery of the message. Here we show that depending on the mRNA loading level, LNPs exhibit distinct mass densities and can be fractionated via ultracentrifugation.

Genetic engineering of megakaryocytes from blood progenitor cells using messenger RNA lipid nanoparticles.

Background: Platelets are an essential component of hemorrhage control and management, and engineering platelets to express therapeutic proteins could expand their use as a cell therapy. Genetically engineered platelets can be achieved by modifying the platelet precursor cells, megakaryocytes (MKs). Current strategies include transfecting MK progenitors ex vivo with viral vectors harboring lineage-driven transgenes and inducing the production of in vitro modified platelets.

Protein is expressed in all major organs after intravenous infusion of mRNA-lipid nanoparticles in swine.

delivery of mRNA is promising for the study of gene expression and the treatment of diseases. Lipid nanoparticles (LNPs) enable efficient delivery of mRNA constructs, but protein expression has been assumed to be limited to the liver. With specialized LNPs, delivery to extrahepatic tissue occurs in small animal models; however, it is unclear if global delivery of mRNA to all major organs is possible in humans because delivery may be affected by differences in innate immune response and relative organ size.

Genetic engineering of transfusable platelets with mRNA-lipid nanoparticles is compatible with blood banking practices.

Platelets contribute to a variety of physiological processes, including inflammation, sepsis, and cancer. However, because of their primary role in hemostasis, platelet transfusions are largely restricted to managing thrombocytopenia and bleeding. One way to expand the utility of platelet transfusions would be to genetically engineer donor platelets with new or enhanced functions.

The 60-year evolution of lipid nanoparticles for nucleic acid delivery.

Delivery of genetic information to the interior of target cells in vivo has been a major challenge facing gene therapies. This barrier is now being overcome, owing in part to dramatic advances made by lipid-based systems that have led to lipid nanoparticles (LNPs) that enable delivery of nucleic acid-based vaccines and therapeutics. Examples include the clinically approved COVID-19 LNP mRNA vaccines and Onpattro (patisiran), an LNP small interfering RNA therapeutic to treat transthyretin-induced amyloidosis (hATTR).

siRNA-mediated reduction of a circulating protein in swine using lipid nanoparticles.

Genetic manipulation of animal models is a fundamental research tool in biology and medicine but is challenging in large animals. In rodents, models can be readily developed by knocking out genes in embryonic stem cells or by knocking down genes through delivery of nucleic acids. Swine are a preferred animal model for studying the cardiovascular and immune systems, but there are limited strategies for genetic manipulation.

Direct in vivo CAR T cell engineering.

T cells modified to express intelligently designed chimeric antigen receptors (CARs) are exceptionally powerful therapeutic agents for relapsed and refractory blood cancers and have the potential to revolutionize therapy for many other diseases. To circumvent the complexity and cost associated with broad-scale implementation of ex vivo manufactured adoptive cell therapy products, alternative strategies to generate CAR T cells in vivo by direct infusion of nanoparticle-formulated nucleic acids or engineered viral vectors under development have received a great deal of attention in the past few years. Here, we outline the ex vivo manufacturing process as a motivating framework for direct in vivo strategies and discuss emerging data from preclinical models to highlight the potency of the in vivo approach, the applicability for new disease indications, and the remaining challenges associated with clinical readiness, including delivery specificity, long term efficacy, and safety.

A magnetic separation method for isolating and characterizing the biomolecular corona of lipid nanoparticles.

Lipid nanoparticle (LNP) formulations are a proven method for the delivery of nucleic acids for gene therapy as exemplified by the worldwide rollout of LNP-based RNAi therapeutics and mRNA vaccines. However, targeting specific tissues or cells is still a major challenge. After LNP administration, LNPs interact with biological fluids (i.

Lipid nanoparticles and siRNA targeting plasminogen provide lasting inhibition of fibrinolysis in mouse and dog models of hemophilia A.

Antifibrinolytic drugs are used extensively for on-demand treatment of severe acute bleeding. Controlling fibrinolysis may also be an effective strategy to prevent or lessen chronic recurring bleeding in bleeding disorders such as hemophilia A (HA), but current antifibrinolytics have unfavorable pharmacokinetic profiles. Here, we developed a long-lasting antifibrinolytic using small interfering RNA (siRNA) targeting plasminogen packaged in clinically used lipid nanoparticles (LNPs) and tested it to determine whether reducing plasmin activity in animal models of HA could decrease bleeding frequency and severity.

Determination of the interior pH of lipid nanoparticles using a pH-sensitive fluorescent dye-based DNA probe.

Lipid nanoparticles (LNPs) containing ionizable cationic lipids are proven delivery systems for therapeutic nucleic acids, such as small interfering RNA (siRNA). It is important to understand the relationship between the interior pH of LNPs and the pH of the external environment to understand LNP formulation and function. Here, we developed a simple and rapid approach for determining the pH of the LNP core using a pH-sensitive fluorescent dye-based DNA probe.

Considerations on the Design of Lipid-based mRNA Vaccines Against Cancer.

Throughout the last decades, mRNA vaccines have been developed as a cancer immunotherapeutic and the technology recently gained momentum during the COVID-19 pandemic. Recent promising results obtained from clinical trials investigating lipid-based mRNA vaccines in cancer therapy further highlighted the potential of this therapy. Interestingly, while the technologies being used in authorized mRNA vaccines for the prevention of COVID-19 are relatively similar, mRNA vaccines in clinical development for cancer vaccination show marked differences in mRNA modification, lipid carrier, and administration route.

Genetically engineered transfusable platelets using mRNA lipid nanoparticles.

Platelet transfusions are essential for managing bleeding and hemostatic dysfunction and could be expanded as a cell therapy due to the multifunctional role of platelets in various diseases. Creating these cell therapies will require modifying transfusable donor platelets to express therapeutic proteins. However, there are currently no appropriate methods for genetically modifying platelets collected from blood donors.

Single Pulse Nanosecond Laser-Stimulated Targeted Delivery of Anti-Cancer Drugs from Hybrid Lipid Nanoparticles Containing 5 nm Gold Nanoparticles.

Encapsulating chemotherapeutic drugs like doxorubicin (DOX) inside lipid nanoparticles (LNPs) can overcome their acute, systematic toxicity. However, a precise drug release at the tumor microenvironment for improving the maximum tolerated dose and reducing side effects has yet to be well-established by implementing a safe stimuli-responsive strategy. This study proposes an integrated nanoscale perforation to trigger DOX release from hybrid plasmonic multilamellar LNPs composed of 5 nm gold (Au) NPs clustered at the internal lamellae interfaces.

Lipid Nanoparticle-Mediated Hit-and-Run Approaches Yield Efficient and Safe Gene Editing in Human Skin.

Despite exciting advances in gene editing, the efficient delivery of genetic tools to extrahepatic tissues remains challenging. This holds particularly true for the skin, which poses a highly restrictive delivery barrier. In this study, we ran a head-to-head comparison between Cas9 mRNA or ribonucleoprotein (RNP)-loaded lipid nanoparticles (LNPs) to deliver gene editing tools into epidermal layers of human skin, aiming for gene editing.

Lipid nanoparticle mRNA systems containing high levels of sphingomyelin engender higher protein expression in hepatic and extra-hepatic tissues.

Lipid nanoparticles (LNPs) for delivery of mRNA usually contain ionizable lipid/helper lipid/cholesterol/PEG-lipid in molar ratios of 50:10:38.5:1.5, respectively.

Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency.

The transfection potency of lipid nanoparticle (LNP) mRNA systems is critically dependent on the ionizable cationic lipid component. LNP mRNA systems composed of optimized ionizable lipids often display distinctive mRNA-rich "bleb" structures. Here, it is shown that such structures can also be induced for LNPs containing nominally less active ionizable lipids by formulating them in the presence of high concentrations of pH 4 buffers such as sodium citrate, leading to improved transfection potencies both in vitro and in vivo.

Continuous freeze-drying of messenger RNA lipid nanoparticles enables storage at higher temperatures.

Messenger RNA (mRNA) lipid nanoparticles (LNPs) have emerged at the forefront during the COVID-19 vaccination campaign. Despite their tremendous success, mRNA vaccines currently require storage at deep freeze temperatures which complicates their storage and distribution, and ultimately leads to lower accessibility to low- and middle-income countries. To elaborate on this challenge, we investigated freeze-drying as a method to enable storage of mRNA LNPs at room- and even higher temperatures.

Light-Activated siRNA Endosomal Release (LASER) by Porphyrin Lipid Nanoparticles.

Lipid nanoparticles (LNPs) have achieved clinical success in delivering small interfering RNAs (siRNAs) for targeted gene therapy. However, endosomal escape of siRNA into the cytosol remains a fundamental challenge for LNPs. Herein, we report a strategy termed light-activated siRNA endosomal release (LASER) to address this challenge.

Morphological Behavior of Liposomes and Lipid Nanoparticles.

Liposomes, which consist of bilayer lipids surrounding interior aqueous compartment(s), were first characterized nearly 60 years ago. Remarkably, many fundamental properties of liposomes and their micellar-like "solid core" counterparts (a lipid monolayer surrounding a hydrophobic core) and transitions between these structures remain poorly understood. In this work, we examine the effects of basic variables on the morphology adopted by lipid-based systems produced by rapid mixing of lipids in ethanol with aqueous media.

On the Influence of Nucleic Acid Backbone Modifications on Lipid Nanoparticle Morphology.

Nucleic acid therapeutics represent a major advance toward treating diseases at their root cause. However, nucleic acids are prone to degradation by serum endonucleases, clearance through the immune system, and rapid degradation in complex medium. To overcome these barriers, nucleic acids frequently include chemical modifications to improve stability or decrease immune responses.

Hepatocyte-targeted delivery of imiquimod reduces hepatitis B virus surface antigen.

Hepatitis B virus (HBV) can rapidly replicate in the hepatocytes after transmission, leading to chronic hepatitis, liver cirrhosis and eventually hepatocellular carcinoma. Interferon-α (IFN-α) is included in the standard treatment for chronic hepatitis B (CHB). However, this therapy causes serious side effects.

Development of a child-friendly oral drug formulation using liposomal multilamellar vesicle technology.

Many medicines are only available in solid dosage forms suitable for adults, and extemporaneous compounding is required to prepare formulations for children. However, this common practice often results in inaccurate dosing and unpleasant taste, reducing the medication adherence. Here, we report the development of a new method to prepare and compound child-friendly oral formulations based on a liposomal multilamellar vesicle (MLV) platform.

Lipid nanoparticles to silence androgen receptor variants for prostate cancer therapy.

Advanced-stage prostate cancer remains an incurable disease with poor patient prognosis. There is an unmet clinical need to target androgen receptor (AR) splice variants, which are key drivers of the disease. Some AR splice variants are insensitive to conventional hormonal or androgen deprivation therapy due to loss of the androgen ligand binding domain at the C-terminus and are constitutively active.

Lipid nanoparticle-mediated silencing of osteogenic suppressor GNAS leads to osteogenic differentiation of mesenchymal stem cells in vivo.

Approved drugs for the treatment of osteoporosis can prevent further bone loss but do not stimulate bone formation. Approaches that improve bone density in metabolic diseases are needed. Therapies that take advantage of the ability of mesenchymal stem cells (MSCs) to differentiate into various osteogenic lineages to treat bone disorders are of particular interest.