Emerging Nucleic Acid Cargos for Next-Generation RNA Vaccines and Therapeutics.

Messenger ribonucleic acid (mRNA)‐based therapies, including conventional linear mRNA (linRNA), circular RNA (circRNA), and self‐amplifying RNA (saRNA), are being developed not only for vaccination but also for protein replacement, gene editing, and regenerative medicine. However, these mRNA modalities differ in structure and function, and their interactions with current non‐viral delivery systems influence their therapeutic efficacy. Here, the in vivo expression kinetics of linRNA, circRNA, and saRNA delivered via lipid nanoparticles (LNPs) or bioreducible poly(cystamine bisacrylamide‐co‐4‐amino‐1‐butanol) (pABOL) polymer are systematically evaluated. At 0.5 µg, Venezuelan equine encephalitis virus (VEEV)‐based saRNA resulted in higher total luciferase expression than 5 µg of linRNA or circRNA highlighting its superior potency. LNPs significantly enhanced expression of non‐amplifying mRNAs compared to pABOL, whereas pABOL delivery of saRNA yielded a ∼2‐fold improvement over LNPs. Furthermore, saRNAs derived from New World alphaviruses expressed 2–6 times more protein than Old World saRNAs when delivered with LNPs; these differences are not observed with pABOL. These findings demonstrate that mRNA modality, saRNA genotype, and delivery platform interact to determine therapeutic protein output. This study provides actionable insights for optimizing mRNA‐based therapeutics across diverse clinical applications.

Nanomaterials for Therapeutic RNA Delivery

Advanced Nanosystems for Clinical Translation

RNA Therapeutics: Top 5 RNA Therapeutics Companies to Watch in 2022

ConspectusThe clinical use of mRNA COVID-19 vaccines developed by Moderna and Pfizer-BioNTech has highlighted the critical role of ionizable lipid nanoparticles (LNPs) in the efficient loading, intracellular delivery, and cytoplasmic release of mRNAs. These LNPs typically comprise an ionizable lipid, a helper lipid, cholesterol, and a PEGylated-lipid, each contributing to the stability, structure, encapsulation efficiency, and nanoparticle-biology interactions of the final mRNA-LNPs both in vitro and in vivo. Notably, the ionizable amino-lipids, ALC-0315 used in the BioNTech/Pfizer vaccine and SM-102 in the Moderna vaccine, possess similar molecular structures, featuring multiple saturated aliphatic chains linked to a tertiary amine group via ester bonds. The acidification-induced ionization behavior of these amino-lipids is essential for enabling endosomal escape and facilitating the intracellular transfection of therapeutic mRNAs. However, despite their widespread clinical use, the physicochemical property-biological interaction and function relationships for LNPs remain poorly understood, particularly regarding how the internal nanostructural evolution during endosomal maturation influences mRNA release, endosomal escape, and gene expression. The rudimentary understanding continues to impede the rational design and optimization of RNA therapeutics.With long-standing expertise in amphiphile self-assembly and structural characterization, especially inverse lyotropic liquid crystalline mesophase-forming lipids, our group seeks to address this critical knowledge gap by establishing a clear connection between pH-triggered mesophase transitions and the biological performance of mRNA-LNPs, with the aim of providing new mechanistic insight into how the internal nanostructure affects mRNA delivery efficiency. This Account focuses on the pH-dependent inverse mesostructural behavior of ionizable LNPs containing two COVID-19 mRNA vaccine ionizable lipids, ALC-0315 and SM-102. We have applied high-throughput and cutting-edge time-resolved synchrotron radiation small-angle X-ray scattering (SAXS) to investigate both static and kinetic self-assembly and structural transitions of these ionizable LNPs without and with nucleic acid cargos (including mRNAs, polyA tails, and plasmid DNAs) upon acidification. We further explored the influence of other components in LNPs, such as select structure-forming helper lipids (monoolein and phytantriol) and cholesterol, on their physicochemical properties, mesophase behavior, and gene delivery performance. Notably, we correlated the mesophase transition of LNPs, from nonordered state to ordered inverse micellar, hexagonal, and cubic phases, with their mRNA transfection efficiency in macrophage cells, providing mechanistic insight into the role of internal nanostructure in endosomal escape and gene expression. Moreover, we addressed the impact of protein coronas formed upon exposure to biological environments, which can significantly alter the LNP internal structure and delivery efficiency. Our findings suggest that protein corona-modulated phase behavior of LNPs may contribute to reported inconsistency between in vitro and in vivo performance. Finally, we offer a perspective on future research trends in improving endosomal escape efficiency, promoting a passive nonendocytic cellular uptake pathway, modulating protein corona effects, monitoring the immune compatibility of PEG-free stabilizers, and leveraging of artificial intelligence approaches to accelerate formulation design and screening. Overall, this Account provides guidance for future mechanistic research with respect to LNP internal structures under various environmental and biological conditions, enabling the rational design of next-generation RNA therapeutics.

Aminophosphonate-Derived Lipid Nanoparticles Enable Circular RNA Delivery for Functional Recovery after Spinal Cord Injury.

Messenger RNA (mRNA), which is composed of ribonucleotides that carry genetic information and direct protein synthesis, is transcribed from a strand of DNA as a template. On this basis, mRNA technology can take advantage of the body’s own translation system to express proteins with multiple functions for the treatment of various diseases. Due to the advancement of mRNA synthesis and purification, modification and sequence optimization technologies, and the emerging lipid nanomaterials and other delivery systems, mRNA therapeutic regimens are becoming clinically feasible and exhibit significant reliability in mRNA stability, translation efficiency, and controlled immunogenicity. Lipid nanoparticles (LNPs), currently the leading non-viral delivery vehicles, have made many exciting advances in clinical translation as part of the COVID-19 vaccines and therefore have the potential to accelerate the clinical translation of gene drugs. Additionally, due to their small size, biocompatibility, and biodegradability, LNPs can effectively deliver nucleic acids into cells, which is particularly important for the current mRNA regimens. Therefore, the cutting-edge LNP@mRNA regimens hold great promise for cancer vaccines, infectious disease prevention, protein replacement therapy, gene editing, and rare disease treatment. To shed more lights on LNP@mRNA, this paper mainly discusses the rational of choosing LNPs as the non-viral vectors to deliver mRNA, the general rules for mRNA optimization and LNP preparation, and the various parameters affecting the delivery efficiency of LNP@mRNA, and finally summarizes the current research status as well as the current challenges. The latest research progress of LNPs in the treatment of other diseases such as oncological, cardiovascular, and infectious diseases is also given. Finally, the future applications and perspectives for LNP@mRNA are generally introduced.

Bend Bioscience invests in expansion of drug-delivery capabilities Bend Bioscience (OR, USA) and NovaQuest Private Equity, have announced plans on 11 October 2022 to invest in a new facility in Bend, Oregon. The group will focus on developing new R&D partnerships, and early phase manufacturing and development activities based on proprietary particle engineering-based drug-delivery technologies. This new facility will consist of approximately 20,000 square feet of laboratory and processing areas and it is anticipated that manufacturing and analytical capabilities will operational by the end of Q2 2023. Announcing the investment decision, Dan Dobry, Bend Bioscience's co-founder said "We feel Bend Bioscience will fill an unmet need, offering problem solving, formulation, and manufacturing for valuable but challenging therapies and collaborating to develop new technologies for the rapidly evolving problem statements in the in pharmaceutical industry" [1].

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.

Cell-specific mRNA delivery via nanobody-functionalized lipid nanoparticles.

mRNA-based gene editing platforms have tremendous promise in the treatment of genetic diseases. However, for this potential to be realized in vivo, these nucleic acid cargos must be delivered safely and effectively to cells of interest. Ionizable lipid nanoparticles (LNPs), the most clinically advanced non-viral RNA delivery system, have been well-studied for the delivery of mRNA but have not been systematically optimized for the delivery of mRNA-based CRISPR-Cas9 platforms. In this study, we investigated the effect of microfluidic and lipid excipient parameters on LNP gene editing efficacy. Through in vitro screening in liver cells, we discovered distinct trends in delivery based on phospholipid, cholesterol, and lipid-PEG structure in LNP formulations. Combination of top-performing lipid excipients produced an LNP formulation that resulted in 3-fold greater gene editing in vitro and facilitated 3-fold greater reduction of a therapeutically-relevant protein in vivo relative to the unoptimized LNP formulation. Thus, systematic optimization of LNP formulation parameters revealed a novel LNP formulation that has strong potential for delivery of gene editors to the liver to treat metabolic disease.

Since the U.S. Food and Drug Administration (FDA) granted emergency use authorization for two mRNA vaccines against SARS-CoV-2, mRNA-based technology has attracted broad attention from the scientific community to investors. When delivered intracellularly, mRNA has the ability to produce various therapeutic proteins, enabling the treatment of a variety of illnesses, including but not limited to infectious diseases, cancers, and genetic diseases. Accordingly, mRNA holds significant therapeutic potential and provides a promising means to target historically hard-to-treat diseases. Current clinical efforts harnessing mRNA-based technology are focused on vaccination, cancer immunotherapy, protein replacement therapy, and genome editing. The clinical translation of mRNA-based technology has been made possible by leveraging nanoparticle delivery methods. However, the application of mRNA for therapeutic purposes is still challenged by the need for specific, efficient, and safe delivery systems.This Account highlights key advances in designing and developing combinatorial synthetic lipid nanoparticles (LNPs) with distinct chemical structures and properties for in vitro and in vivo intracellular mRNA delivery. LNPs represent the most advanced nonviral nanoparticle delivery systems that have been extensively investigated for nucleic acid delivery. The aforementioned COVID-19 mRNA vaccines and one LNP-based small interfering RNA (siRNA) drug (ONPATTRO) have received clinical approval from the FDA, highlighting the success of synthetic ionizable lipids for in vivo nucleic acid delivery. In this Account, we first summarize the research efforts from our group on the development of bioreducible and biodegradable LNPs by leveraging the combinatorial chemistry strategy, such as the Michael addition reaction, which allows us to easily generate a large set of lipidoids with diverse chemical structures. Next, we discuss the utilization of a library screening strategy to identify optimal LNPs for targeted mRNA delivery and showcase the applications of the optimized LNPs in cell engineering and genome editing. Finally, we outline key challenges to the clinical translation of mRNA-based therapies and propose an outlook for future directions of the chemical design and optimization of LNPs to improve the safety and specificity of mRNA drugs. We hope this Account provides insight into the rational design of LNPs for facilitating the development of mRNA therapeutics, a transformative technology that promises to revolutionize future medicine.

The advent of high throughput technologies has revealed that mammalian genomes are pervasively transcribed, most for long noncoding RNAs (lncRNAs, at least 200 nt long). Thousands of lncRNAs from intergenic regions (large intergenic noncoding RNA, lincRNA) have been uncovered by massive deep sequencing from the repertoire of polyadenylated (poly(A)+) RNAs, together with multiple chromatin landscapes. These lncRNAs are messenger RNA (mRNA)-like, with linear signatures of 5′ mG caps and 3′ poly(A)+ tails. Unexpectedly, mammalian transcriptomes are even more complex with the expression of RNAs without polyadenylated tails (poly(A)– RNAs) [1], leading to the identification of new lncRNA formats, such as circular RNAs. Due to the covalently close structure and without 3′ poly(A) tails, circular RNAs failed to be analyzed in most transcriptome analyses mainly for polyadenylated RNAs. By taking advantage of deep sequencing from nonpolyadenylated RNA population [1], thousands of circular RNAs were identified to be widely expressed in human cell lines. There are at least two different types of circular RNAs processed from pre-RNA splicing: one type is derived from spliced introns (circular intronic RNAs) [2] and the other type is from back-spliced exons (exonic circular RNAs) [3]. Circular intronic RNAs (ciRNAs) are produced from introns that fail to be debranched after splicing, but covalently circularized with 2′,5′-phosphodiester bond between a splice donor site and a branch point site. The formation of ciRNAs can be reconstituted in expression vectors with the requirement of consensus motifs flanking 2′,5′-phosphodiester bonds. Importantly, ciRNAs were shown to play an important cis-regulatory role in local gene expression [2]. Exonic circular RNAs (circRNAs) are produced from back-spliced circularization [3]. Unlike (normal) RNA splicing that joins an upstream splice donor site with a downstream splice acceptor site, leading to a linear RNA transcript (Figure 1A), back splicing joins a downstream splice donor site reversely with an upstream splice acceptor site, yielding a circular RNA transcript with 3′,5′-phosphodiester bond at the joint site (Figure 1B). In last decades, only a handful of circRNAs were identified and indicated as byproducts of splicing errors with no function. Until recently, the genome-wide profiling of

The advent of messenger RNA (mRNA) therapeutics has revolutionized medicine, with its potential underscored by rapid advancements during the COVID-19 pandemic. Despite its promise, nucleic acid delivery remains a formidable challenge due to enzymatic degradation, cellular uptake barriers, and endosomal trapping. Therapeutic lipid nanoparticles (LNPs), pioneered in the 1970s, have emerged as the gold standard for delivering mRNA and other nucleic acids, offering unparalleled advantages in stability, biocompatibility, and cellular targeting. This review explores the evolution and design of LNPs, focusing on their role in hematologic therapies and platelet transfection, where unique challenges arise due to platelets' anucleate nature. The paper systematically evaluates the composition of LNPs, highlighting the role of ionizable, cationic, and neutral lipids in optimizing delivery efficiency, stability, and immune response modulation. Strategies to overcome platelet transfection barriers, including tailored lipid compositions and particle engineering, are discussed alongside advances in artificial intelligence (AI) for predictive nanoparticle design. Furthermore, it examines various nucleic acid cargoes, including mRNA, siRNA, and miRNA, and their therapeutic potential in addressing platelet-related disorders and advancing personalized medicine. Finally, the review delves into emerging technologies and the integration of AI to overcome existing barriers in nucleic acid delivery. By fostering interdisciplinary collaboration, this work aims to catalyze discoveries in LNP-based therapeutics and transformative advancements in hematologic treatments.

Ionizable lipid nanoparticles encapsulating barcoded mRNA for accelerated in vivo delivery screening