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Genetic drugs such as small interfering RNA (siRNA), mRNA, or plasmid DNA provide potential gene therapies to treat most diseases by silencing pathological genes, expressing therapeutic proteins, or through gene-editing applications. In order for genetic drugs to be used clinically, however, sophisticated delivery systems are required. Lipid nanopa...
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... we focus on nonviral delivery systems that have advantages of ease of manufacture, reduced immune responses, multi-dosing capabilities, larger payloads, and flexibility of design. The lead non-viral delivery systems are lipid nanoparticles (LNPs); more than four LNP small interfering RNA (siRNA) drugs have entered the clinic, one of which is in late stage phase III trials (see Table 1). Here, we describe the origins of LNP technology for delivery of small molecule drugs, summarize the developments leading to encapsulation and delivery of genetic drugs, and indicate the ongoing optimization process 3 that is leading to increasingly potent, non-toxic gene therapies with clear clinical potential. ...Citations
... Despite that researchers have most recently reported that iPLX phospholipid-free LNP systems presented exceptional stability [28], superior mRNA encapsulation efficiency, and sustained robust delivery efficacy, the three FDA-approved LNP-RNA products as well as most LNP-mRNA in preclinical reports conventionally are constituted of four components: ionizable or cationic lipids, sterols, helper lipids, and PEGylated lipids. To better illustrate the conceptions, the lipids mentioned in this review were demonstrated in Fig. 2. Cationic or ionizable lipids, such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) or clinically approved DLin-MC3-DMA, primarily interact with negatively charged mRNA backbone to form electrostatic bonds, thus stabilizing the particles with enhancing encapsulation efficiency as well as the transduction efficacy [17,26,29,30]. Sterols, mostly present as cholesterol, are in charge of securing the particle's formation as a building block supplement. ...
Messenger RNA (mRNA) has emerged as an innovative therapeutic modality, offering promising avenues for the prevention and treatment of a variety of diseases. The tremendous success of mRNA vaccines in effectively combatting coronavirus disease 2019 (COVID-19) evidences the unlimited medical and therapeutic potential of mRNA technology. Overcoming challenges related to mRNA stability, immunogenicity, and precision targeting has been made possible by recent advancements in lipid nanoparticles (LNPs). This review summarizes state-of-the-art LNP-mRNA-based therapeutics, including their structure, material compositions, design guidelines, and screening principles. Additionally, we highlight current preclinical and clinical trends in LNP-mRNA therapeutics in a broad range of treatments in ophthalmological conditions, cancer immunotherapy, gene editing, and rare-disease medicine. Particular attention is given to the translation and evolution of LNP-mRNA vaccines into a broader spectrum of therapeutics. We explore concerns in the aspects of inadequate extrahepatic targeting efficacy, elevated doses, safety concerns, and challenges of large-scale production procedures. This discussion may offer insights and perspectives on near- and long-term clinical development prospects for LNP-mRNA therapeutics.
... Lipid nanoparticles (LNPs), with their core component consisting of cationic or ionizable lipids, are employed to package negatively charged nucleic acids [1][2][3][4][5]. Ionizable lipids containing secondary and/or tertiary amines are advantageous due to chemical architecture flexibility, providing endosomal pH-specific responsiveness [6][7][8][9]. ...
Insufficient endosomal escape presents a major hurdle for successful nucleic acid therapy. Here, for the first time, a chemical electron transfer (CET) system was integrated into small interfering RNA (siRNA) lipid nanoparticles (LNPs). The CET acceptor can be chemically excited using the generated energy between the donor and hydrogen peroxide, which triggers the generation of reactive oxygen species (ROS), promoting endosomal lipid membrane destabilization. Tetra-oleoyl tri-lysino succinoyl tetraethylene pentamine was included as an ionizable lipopeptide with a U-shaped topology for effective siRNA encapsulation and pH-induced endosomal escape. LNPs loaded with siRNA and CET components demonstrated a more efficient endosomal escape, as evidenced by a galectin-8-mRuby reporter; ROS significantly augmented galectin-8 recruitment by at least threefold compared with the control groups, with a p value of 0.03. Moreover, CET-enhanced LNPs achieved a 24% improvement in apoptosis level by knocking down the tumor-protective gene nuclear factor erythroid 2-related factor 2, boosting the CET-mediated ROS cell killing.
... 1,2) Since the development and manufacturing of vaccines or therapeutics based on this modality is much faster than that of other modality platforms, many mRNA candidates have been developed for future pandemics, cancer vaccines, and disease therapeutics. 3) Breakthroughs in the medical application of mRNA were brought about by the development of encapsulation techniques for delivery, and lipid nanoparticles (LNPs) have emerged as the leading technology for nucleic acid delivery. [4][5][6][7][8] LNPs are typically composed of ionizable lipids, phospholipids, cholesterol, and lipid-anchored polyethylene glycol (PEG), with ionizable lipids being most important for protein expression by mRNA. ...
Lipid nanoparticles (LNPs), used for mRNA vaccines against severe acute respiratory syndrome coronavirus 2, protect mRNA and deliver it into cells, making them an essential delivery technology for RNA medicine. The LNPs manufacturing process consists of two steps, the upstream process of preparing LNPs and the downstream process of removing ethyl alcohol (EtOH) and exchanging buffers. Generally, a microfluidic device is used in the upstream process, and a dialysis membrane is used in the downstream process. However, there are many parameters in the upstream and downstream processes, and it is difficult to determine the effects of variations in the manufacturing parameters on the quality of the LNPs and establish a manufacturing process to obtain high-quality LNPs. This study focused on manufacturing mRNA-LNPs using a microfluidic device. Extreme gradient boosting (XGBoost), which is a machine learning technique, identified EtOH concentration (flow rate ratio), buffer pH, and total flow rate as the process parameters that significantly affected the particle size and encapsulation efficiency. Based on these results, we derived the manufacturing conditions for different particle sizes (approximately 80 and 200 nm) of LNPs using Bayesian optimization. In addition, the particle size of the LNPs significantly affected the protein expression level of mRNA in cells. The findings of this study are expected to provide useful information that will enable the rapid and efficient development of mRNA-LNPs manufacturing processes using microfluidic devices.
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... Additionally, LNPs were approved as hepatocytetargeted siRNA drug Onpattro for treatment of hereditary transthyretin amyloidosis [3]. The chemical space of synthetic carriers [4,5] ranges from small cationic lipids applied in lipoplexes [6][7][8][9][10] and lipid nanoparticles (LNPs) [3,[10][11][12][13][14][15][16][17], over medium-sized sequence-defined xenopeptides to macromolecular polycations applied in polyplexes and polymer micelles [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36]. In general, the efficacies of synthetic vectors are still moderate on nanoparticle basis, but can be compensated by enhanced nanoparticle dose, as well tolerated formulations can be produced in precise form and at large scale. ...
... Such targeted delivery minimizes the risk of systemic side effects and enhances therapeutic efficacy [7]. Storage-wise, lipid-based nanoparticles exhibit superior stability compared to other drug delivery systems [8]. They can withstand temperature and pH variations, ensuring sustained drug efficiency over time [9]. ...
Lipid-based nanoparticles hold great potential for drug delivery, providing biocompatibility and the ability to encapsulate both hydrophilic and hydrophobic drugs. However, there are certain challenges associated with small molecules, such as leakage and premature release, which can compromise their effectiveness. Despite these challenges, lipid nanoparticles offer advantages in terms of solubility, stability, and targeted delivery, thereby reducing side effects. Additionally, they can be customized for specific molecules, ensuring biocompatibility and biodegradability. While complications may arise, lipid nanoparticles offer numerous benefits for loading biomolecules, improving pharmacokinetics, and enhancing therapeutic effects. It is important to address stability and loading challenges when encapsulating biomolecules and consider potential immunogenic responses that may impact biocompatibility and safety.
... By showing the occurrence of two essential proinflammatory effects by mRNA-LNP vaccines, the present study attempts to give a scientific explanation for the "special interest" SAEs that these vaccines rarely cause, yet that induce substantial press and media attention with top-level public debates in the U.S. Senate and U.K. Parliament in the context of vaccine safety and excess death. The number of publications on different COVID-19 vaccine-induced SAEs has been on the rise , and once the real scale and mechanism of these AEs are better understood, the focus can be redirected to the prevention and, hence, further extension of mRNA-LNP technology for the development of new vaccines and gene therapies [78][79][80]. The present study contributes to the latter goal. ...
A small fraction of people vaccinated with mRNA–lipid nanoparticle (mRNA-LNP)-based COVID-19 vaccines display acute or subacute inflammatory symptoms whose mechanism has not been clarified to date. To better understand the molecular mechanism of these adverse events (AEs), here, we analyzed in vitro the vaccine-induced induction and interrelations of the following two major inflammatory processes: complement (C) activation and release of proinflammatory cytokines. Incubation of Pfizer-BioNTech’s Comirnaty and Moderna’s Spikevax with 75% human serum led to significant increases in C5a, sC5b-9, and Bb but not C4d, indicating C activation mainly via the alternative pathway. Control PEGylated liposomes (Doxebo) also induced C activation, but, on a weight basis, it was ~5 times less effective than that of Comirnaty. Viral or synthetic naked mRNAs had no C-activating effects. In peripheral blood mononuclear cell (PBMC) cultures supplemented with 20% autologous serum, besides C activation, Comirnaty induced the secretion of proinflammatory cytokines in the following order: IL-1α < IFN-γ < IL-1β < TNF-α < IL-6 < IL-8. Heat-inactivation of C in serum prevented a rise in IL-1α, IL-1β, and TNF-α, suggesting C-dependence of these cytokines’ induction, although the C5 blocker Soliris and C1 inhibitor Berinert, which effectively inhibited C activation in both systems, did not suppress the release of any cytokines. These findings suggest that the inflammatory AEs of mRNA-LNP vaccines are due, at least in part, to stimulation of both arms of the innate immune system, whereupon C activation may be causally involved in the induction of some, but not all, inflammatory cytokines. Thus, the pharmacological attenuation of inflammatory AEs may not be achieved via monotherapy with the tested C inhibitors; efficacy may require combination therapy with different C inhibitors and/or other anti-inflammatory agents.
... Continuous PE evolution in OrthoRep could potentially lead to variants with higher activity than PE_Y18. This would be desired for the clinical translation of prime editing, since in vivo genome editing rates with PE_Y18 did not reach levels that are typically achieved with Cas9 nucleases or base editors 2 , and AAV doses above what would be deemed safe for human application 28,29 had to be used. Another limitation for efficient prime editing is the stability of the pegRNA, particularly the 3'end, which is not protected by Cas9 and readily degraded by exo-and endonucleases. ...
Prime editing is a highly versatile genome editing technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, a yeast-based platform for directed protein evolution, to enhance the editing efficiency of PEs. After several rounds of evolution with increased selection pressure, we identify multiple mutations that have a positive effect on PE activity in yeast cells and in biochemical assays. Combining the two most effective mutations – the A259D amino acid substitution in nCas9 and the K445T substitution in M-MLV RT – results in the variant PE_Y18. Delivery of PE_Y18, encoded on DNA, mRNA or as a ribonucleoprotein complex into mammalian cell lines increases editing rates up to 3.5-fold compared to PEmax. In addition, PE_Y18 supports higher prime editing rates when delivered in vivo into the liver or brain. Our study demonstrates proof-of-concept for the application of OrthoRep to optimize genome editing tools in eukaryotic cells.
... Initial lipoplexes (24,25) were designed as complexes of cationic lipids and nucleic acid, stabilizing and compacting the nucleic acid cargo into nanoparticles by electrostatic and hydrophobic forces (26). In LNPs (27)(28)(29)(30), combination of the cationic lipid with cholesterol, phospholipids, and a polyethylene glycol (PEG)-lipid merges the advantages of lipoplexes and liposomes: robust encapsulation of RNA into nanoparticles by artificial cationic lipids on the one hand, complemented by the properties of natural lipid membrane forming components and protective PEG-lipid on the other hand, and optionally additional modifications for chemical or ligand targeting. Conceptual benefits of LNPs include their controlled self-assembly in well-established production processes based on low-molecular weight natural or artificial lipid components. ...
... As multicomponent assemblies, LNPs can be improved by combinatorial variation of phospholipids, PEG-lipid, cholesterol analogs, and/or the ionizable lipid. Optimizing the cationizable lipid structure (Fig. 1A) by modifying hydrophilic head group and hydrophobic tails, optionally including degradable bonds, was key for the LNP efficacy (27,(36)(37)(38). First breakthrough was the replacement of permanent cationic lipids such as DOTMA or DOTAP by ionizable tertiary amine-containing lipids. ...
Carriers for RNA delivery must be dynamic, first stabilizing and protecting therapeutic RNA during delivery to the target tissue and across cellular membrane barriers and then releasing the cargo in bioactive form. The chemical space of carriers ranges from small cationic lipids applied in lipoplexes and lipid nanoparticles, over medium-sized sequence-defined xenopeptides, to macromolecular polycations applied in polyplexes and polymer micelles. This perspective highlights the discovery of distinct virus-inspired dynamic processes that capitalize on mutual nanoparticle–host interactions to achieve potent RNA delivery. From the host side, subtle alterations of pH, ion concentration, redox potential, presence of specific proteins, receptors, or enzymes are cues, which must be recognized by the RNA nanocarrier via dynamic chemical designs including cleavable bonds, alterable physicochemical properties, and supramolecular assembly–disassembly processes to respond to changing biological microenvironment during delivery.
... To test whether layering was modular across LNPs, we formulated a set of LNP cores, varying in component lipids, lipid ratios, and nucleic acid cargos (either mRNA or pDNA). Formulations contained commercially available ionizable cationic lipids used in prior applications, with either single valency (DLin-MC3-DMA, DLin-KC2-DMA, and ALC-0315) or multiple valency (C12-200 and cKK-E12) (SI Appendix, Table S1) (2,(27)(28)(29)(30). LNPs were suspended in acidic conditions to express positive surface charge, then incubated in equal volumes of each of the four polyanions (Fig. 1B). ...
Rapid advances in nucleic acid therapies highlight the immense therapeutic potential of genetic therapeutics. Lipid nanoparticles (LNPs) are highly potent nonviral transfection agents that can encapsulate and deliver various nucleic acid therapeutics, including but not limited to messenger RNA (mRNA), silencing RNA (siRNA), and plasmid DNA (pDNA). However, a major challenge of targeted LNP-mediated systemic delivery is the nanoparticles’ nonspecific uptake by the liver and the mononuclear phagocytic system, due partly to the adsorption of endogenous serum proteins onto LNP surfaces. Tunable LNP surface chemistries may enable efficacious delivery across a range of organs and cell types. Here, we describe a method to electrostatically adsorb bioactive polyelectrolytes onto LNPs to create layered LNPs (LLNPs). LNP cores varying in nucleic acid cargo and component lipids were stably layered with four biologically relevant polyanions: hyaluronate (HA), poly-L-aspartate (PLD), poly-L-glutamate (PLE), and polyacrylate (PAA). We further investigated the impact of the four surface polyanions on the transfection and uptake of mRNA- and pDNA-loaded LNPs in cell cultures. PLD- and PLE-LLNPs increased mRNA transfection twofold over unlayered LNPs in immune cells. HA-LLNPs increased pDNA transfection rates by more than twofold in epithelial and immune cells. In a healthy C57BL/6 murine model, PLE- and HA-LLNPs increased transfection by 1.8-fold to 2.5-fold over unlayered LNPs in the liver and spleen. These results suggest that LbL assembly is a generalizable, highly tunable platform to modify the targeting specificity, stability, and transfection efficacy of LNPs, as well as incorporate other charged targeting and therapeutic molecules into these systems.
... The IL plays a vital role in the development of LNP formulations for gene therapy [26][27][28]. One of the primary obstacles in developing LNP is synthesizing and purifying IL with optimal characteristics, such as efficient pKa, low toxicity, proper endosomal escape, and minimal post-formulation residuals [29,30]. ...
The development of ionizable lipid (IL) was necessary to enable the effective formulation of small interfering RNA (siRNA) to inhibit P2X7 receptors (P2X7R), a key player in tumor proliferation, apoptosis, and metastasis. In this way, the synthesis and utility of IL for enhancing cellular uptake of lipid nanoparticles (LNP) improve the proper delivery of siRNA-LNPs for knockdown overexpression of P2X7R. Therefore, to evaluate the impact of P2X7 knockdown on breast cancer (BC) migration and apoptosis, a branched and synthesized ionizable lipid (SIL) was performed for efficient transfection of LNP with siRNA for targeting P2X7 receptors (siP2X7) in mouse 4T-1 cells. Following synthesis and structural analysis of SIL, excellent characterization of the LNP was achieved (Z-average 126.8 nm, zeta-potential − 12.33, PDI 0.16, and encapsulation efficiency 85.35%). Afterward, the stability of the LNP was evaluated through an analysis of the leftover composition, and toxic concentration values for SIL and siP2X7 were determined. Furthermore, siP2X7-LNP cellular uptake in the formulation was assessed via confocal microscopy. Following determining the optimal dose (45 pmol), wound healing analysis was assessed using scratch assay microscopy, and apoptosis was evaluated using flow cytometry. The use of the innovative branched SIL in the formulation of siP2X7-LNP resulted in significant inhibition of migration and induction of apoptosis in 4T-1 cells due to improved cellular uptake. Subsequently, the innovative SIL represents a critical role in efficiently delivering siRNA against murine triple-negative breast cancer cells (TNBC) using LNP formulation, resulting in significant efficacy.
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