704/DNA vaccines leverage cytoplasmic DNA stimulation to promote anti-HIV neutralizing antibody production in mice and strong immune response against alpha-fetoprotein in non-human primates.

Genetic immunization is an attractive approach for prophylactic and therapeutic vaccination using synthetic vectors to deliver antigen-encoding nucleic acids. Recently, DNA delivered by a physical means or RNA by liposomes consisting of four different lipids demonstrated good protection in human phase III clinical trials and received Drugs Controller General of India and US FDA approval to protect against COVID-19, respectively. However, the development of a system allowing for efficient and simple delivery of nucleic acids while improving immune response priming has the potential to unleash the full therapeutic potential of genetic immunization. DNA-based gene therapies and vaccines have the potential for rapid development, as exemplified by the recent approval of Collategene, a gene therapy to treat human critical limb ischemia, and ZyCoV, a DNA vaccine delivered by spring-powered jet injector to protect against SARS-CoV2 infection. Recently, we reported amphiphilic block copolymer 704 as a promising synthetic vector for DNA vaccination in various models of human diseases. This vector allows dose sparing of antigen-encoding plasmid DNA. Here, we report the capacity of 704-mediated HIV and anti-hepatocellular carcinoma DNA vaccines to induce the production of specific antibodies against gp120 HIV envelope proteins in mice and against alpha-fetoprotein antigen in non-human primates, respectively. An investigation of the underlying mechanisms showed that 704-mediated vaccination did trigger a strong immune response by (1) allowing a direct DNA delivery into the cytosol, (2) promoting an intracytoplasmic DNA sensing leading to both interferon and NF-κB cascade stimulation, and (3) inducing antigen expression by muscle cells and presentation by antigen-presenting cells, leading to the induction of a robust adaptive response. Overall, our findings suggest that the 704-mediated DNA vaccination platform is an attractive method to develop both prophylactic and therapeutic vaccines.

Production, Characterization, and Assessment of Permanently Cationic and Ionizable Lipid Nanoparticles for Use in the Delivery of Self-Amplifying RNA Vaccines.

Africa bears the highest burden of infectious diseases, yet the continent is heavily reliant on First World countries for the development and supply of life-saving vaccines. The COVID-19 pandemic was a stark reminder of Africa's vaccine dependence and since then great interest has been generated in establishing mRNA vaccine manufacturing capabilities on the African continent. Herein, we explore alphavirus-based self-amplifying RNAs (saRNAs) delivered by lipid nanoparticles (LNPs) as an alternative to the conventional mRNA vaccine platform. The approach is intended to produce dose-sparing vaccines which could assist resource-constrained countries to achieve vaccine independence. Protocols to synthesize high-quality saRNAs were optimized and in vitro expression of reporter proteins encoded by saRNAs was achieved at low doses and observed for an extended period. Permanently cationic or ionizable LNPs (cLNPs and iLNPs, respectively) were successfully produced, incorporating saRNAs either exteriorly (saRNA-Ext-LNPs) or interiorly (saRNA-Int-LNPs). DOTAP and DOTMA saRNA-Ext-cLNPs performed best and were generally below 200 nm with good PDIs (<0.3). DOTAP and DDA saRNA-Int-cLNPs performed optimally, allowing for saRNA amplification. These were slightly larger, with higher PDIs as a result of the method used, which will require further optimization. In both cases, the N:P ratio and lipid molar ratio had a distinct effect on saRNA expression kinetics, and RNA was encapsulated at high percentages of >90%. These LNPs allow the delivery of saRNA with no significant toxicity. The optimization of saRNA production and identification of potential LNP candidates will facilitate saRNA vaccine and therapeutic development. The dose-sparing properties, versatility, and manufacturing simplicity of the saRNA platform will facilitate a rapid response to future pandemics.

mRNA therapeutics: New vaccination and beyond

The idea of mRNA therapy had been conceived for decades before it came into reality during the Covid-19 pandemic. The mRNA vaccine emerges as a powerful and general tool against new viral infections, largely due to its versatility and rapid development. In addition to prophylactic vaccines, mRNA technology also offers great promise for new applications as a versatile drug modality. However, realizing the conceptual potential faces considerable challenges, such as minimal immune stimulation, high and long-term expression, and efficient delivery to target cells and tissues. Here we review the applications of mRNA-based therapeutics, with emphasis on the innovative design and future challenges/solutions. In addition, we also discuss the next generation of mRNA therapy, including circular mRNA and self-amplifying RNAs. We aim to provide a conceptual overview and outlook on mRNA therapeutics beyond prophylactic vaccines. © 2023

RNA Delivery to Mitochondria.

The approval of mRNA-containing lipid nanoparticles (LNPs) for use in a vaccine against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the clinical utility of RNA-loaded nanocapsules has stimulated a rapid acceleration in research in this area. The development of mRNA-containing LNP vaccines has been rapid, not only because of regulatory adjustments, but also to the advances made in nucleic acid delivery as the result of efforts by many basic researchers. RNA functions, not only in the nucleus and cytoplasm, but also in mitochondria, which have their own genomic apparatus. Mitochondrial diseases caused by mutations or defects in the mitochondrial genome, mitochondrial DNA (mtDNA) are intractable and are mainly treated symptomatically, but gene therapy as a fundamental treatment is expected to soon be a reality. To realize this therapy, a drug delivery system (DDS) that delivers nucleic acids including RNA to mitochondria is required, but efforts in this area have been limited compared to research targeting the nucleus and cytoplasm. This contribution provides an overview of mitochondria-targeted gene therapy strategies and discusses studies that have attempted to validate mitochondria-targeted RNA delivery therapies. We also present the results of 'RNA delivery to mitochondria' based on the use of our mitochondria-targeted DDS (MITO-Porter) that was developed in our laboratory.

Biodrug Delivery Systems: Do mRNA Lipid Nanoparticles Come of Age?

As an appealing alternative to treat and prevent diseases ranging from cancer to COVID-19, mRNA has demonstrated significant clinical effects. Nanotechnology facilitates the successful implementation of the systemic delivery of mRNA for safe human consumption. In this manuscript, we provide an overview of current mRNA therapeutic applications and discuss key biological barriers to delivery and recent advances in the development of nonviral systems. The relevant challenges that LNPs face in achieving cost-effective and widespread clinical implementation when delivering mRNA are likewise discussed.

Optimization of storage conditions for lipid nanoparticle-formulated self-replicating RNA vaccines.

The recent clinical success of multiple mRNA-based SARS-CoV-2 vaccines has proven the potential of RNA formulated in lipid nanoparticles (LNPs) in humans, and products based on base-modified RNA, sequence-optimized RNA, and self-replicating RNAs formulated in LNPs are all in various stages of clinical development. However, much remains to be learned about critical parameters governing the manufacturing and use of LNP-RNA formulations. One important issue that has received limited attention in the literature to date is the identification of optimal storage conditions for LNP-RNA that preserve long-term activity of the formulations. Here, we analyzed the physical structure, in vivo expression characteristics, and functional activity of alphavirus-derived self-replicating RNA (repRNA)-loaded LNPs encoding HIV vaccine antigens following storage in varying temperatures, buffers, and in the presence or absence of cryoprotectants. We found that for lipid nanoparticles with compositions similar to clinically-used LNPs, storage in RNAse-free PBS containing 10% (w/v) sucrose at -20 °C was able to maintain vaccine stability and in vivo potency at a level equivalent to freshly prepared vaccines following 30 days of storage. LNPs loaded with repRNA could also be lyophilized with retention of bioactivity.

Development of mRNA Vaccines/Therapeutics and Their Delivery System.

The rapid development of mRNA vaccines has contributed to the management of the current coronavirus disease 2019 (COVID-19) pandemic, suggesting that this technology may be used to manage future outbreaks of infectious diseases. Because the antigens targeted by mRNA vaccines can be easily altered by simply changing the sequence present in the coding region of mRNA structures, it is more appropriate to develop vaccines, especially during rapidly developing outbreaks of infectious diseases. In addition to allowing rapid development, mRNA vaccines have great potential in inducing successful antigen-specific immunity by expressing target antigens in cells and simultaneously triggering immune responses. Indeed, the two COVID-19 mRNA vaccines approved by the U.S. Food and Drug Administration have shown significant efficacy in preventing infections. The ability of mRNAs to produce target proteins that are defective in specific diseases has enabled the development of options to treat intractable diseases. Clinical applications of mRNA vaccines/therapeutics require strategies to safely deliver the RNA molecules into targeted cells. The present review summarizes current knowledge about mRNA vaccines/ therapeutics, their clinical applications, and their delivery strategies.

RNA/Polymer-Based Supramolecular Approaches for mRNA Delivery

Messenger RNA (mRNA) therapeutics garner growing attention, especially after the approval of two mRNA vaccine formulations for COVID-19. Meanwhile, as a therapeutic modality, mRNA still has issues of poor bioavailability, showing rapid enzymatic degradation in physiological environments and inducing uncontrollable inflammatory responses. Chemical modification of mRNA is a prevalent approach to these issues and effectively reduces mRNA immunogenicity. However, available modified nucleotide species are limited to protect mRNA from enzymatic attacks with preserved mRNA translational activity. Alternatively, in supramolecular approaches, mRNA can be formulated with other molecules to improve its bioavailability. This approach requires minimal modification of mRNA and thus preserves its translational activity. Nano-particulated mRNA formulations using lipids and polymers are widely studied. Among them, polymeric micelles effectively prevent enzymatic mRNA degradation in biological milieu after recent advances in polymer design, allowing safe and efficient mRNA delivery to various organs. Supramolecular approaches also include mRNA formulation using complementary RNA oligonucleotides, which allows the installation of protective moieties to mRNA or crosslinking of several mRNA strands to improve mRNA stability against nucleases. This chapter reviews these strategies to effectively transport mRNA therapeutics to target cells in vivo. © 2022, The Author(s), under exclusive license to Springer Nature Switzerland AG.

A Biopharmaceutical Perspective on Higher-Order Structure and Thermal Stability of mRNA Vaccines.

Preservation of the integrity of macromolecular higher-order structure is a tenet central to achieving biologic drug and vaccine product stability toward manufacturing, distribution, storage, handling, and administration. Given that mRNA lipid nanoparticles (mRNA-LNPs) are held together by an intricate ensemble of weak forces, there are some intriguing parallels to biologic drugs, at least at first glance. However, mRNA vaccines are not without unique formulation and stabilization challenges derived from the instability of unmodified mRNA and its limited history as a drug or vaccine. Since certain learning gained from biologic drug development may be applicable for the improvement of mRNA vaccines, we present a perspective on parallels and contrasts between the emerging role of higher-order structure pertaining to mRNA-LNPs compared to pharmaceutical proteins. In a recent publication, the location of mRNA encapsulated within lipid nanoparticles was identified, revealing new insights into the LNP structure, nanoheterogeneity, and microenvironment of the encapsulated mRNA molecules [Brader et al. Biophys. J. 2021, 120, 2766]. We extend those findings by considering the effect of encapsulation on mRNA thermal unfolding with the observation that encapsulation in LNPs increases mRNA unfolding temperatures.

Recent advances in delivering RNA-based therapeutics to mitochondria.

INTRODUCTION: After the emergence of lipid nanoparticles (LNP) containing therapeutic mRNA as vaccines for use against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the clinical usefulness of nucleic acid-encapsulated LNPs is now a fact. In addition to the nucleus and cytoplasm, mitochondria, which have their own genome, are a site where nucleic acids function in the cell. Gene therapies targeting mitochondria are expected to pave the way for the next generation of therapies. AREAS COVERED: Methods for delivering nucleic acids to mitochondria are needed in order to realize such innovative therapies. However, only a few reports on delivery systems targeting mitochondria have appeared. In this review, we summarize the current state of research on RNA-based therapeutics targeted to mitochondria, with emphasis on mitochondrial RNA delivery therapies and on therapies that involve the use of mitochondrial genome editing devices. EXPERT OPINION: We hope that this review article will focus our attention to this area of research, stimulate more interest in this field of research, and lead to the development of mitochondria-targeted nucleic acid medicine. It has the potential to become a major weapon against urgent and unknown diseases, including SARS-CoV-2 infections.

Lipid Nanoparticles for Drug Delivery

Lipid nanoparticles have attracted significant interests in the last two decades, and have achieved tremendous clinical success since the first clinical approval of Doxil in 1995. At the same time, lipid nanoparticles have also demonstrated enormous potential in delivering nucleic acid drugs as evidenced by the approval of two RNA therapies and mRNA COVID-19 vaccines. In this review, an overview on different classes of lipid nanoparticles, including liposomes, solid lipid nanoparticles, and nanostructured lipid carriers, is first provided, followed by the introduction of their preparation methods. Then the characterizations of lipid nanoparticles are briefly reviewed and their applications in encapsulating and delivering hydrophobic drugs, hydrophilic drugs, and RNAs are highlighted. Finally, various applications of lipid nanoparticles for overcoming different delivery challenges, including crossing the blood-brain barrier, targeted delivery, and various routes of administration, are summarized. Lipid nanoparticles as drug delivery systems offer many attractive benefits such as great biocompatibility, ease of preparation, feasibility of scale-up, nontoxicity, and targeted delivery, while current challenges in drug delivery warrant future studies about structure-function correlations, large-scale production, and targeted delivery to realize the full potential of lipid nanoparticles for wider clinical and pharmaceutical applications in future.

Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery.

In 2021, mRNA vaccines against COVID-19 were approved by the Food and Drug Administration. mRNA vaccines are important for preventing severe COVID-19 and returning to normal life. The development of RNA-delivery technology, including mRNA vaccines, has been investigated worldwide for ~30 years. Lipid nanoparticles (LNPs) are a breakthrough technology that stably delivers RNA to target organs, and RNA-loaded LNP-based nanomedicines have been studied for the development of vaccines and nanomedicines for RNA-, gene-, and cell-based therapies. Recently, microfluidic devices and technologies have attracted attention for the production of LNPs, particularly RNA-loaded LNPs. Microfluidics provides many advantages for RNA-loaded LNP production, including precise LNP size controllability, high reproducibility, high-throughput optimization of LNP formulation, and continuous LNP-production processes. In this review, we summarize microfluidic-based RNA-loaded LNP production and its applications in RNA-based therapy and genome editing.

Recent advances in research on biointerfaces: From cell surfaces to artificial interfaces.

Biointerfaces are regions where biomolecules, cells, and organic materials are exposed to environmental media or come in contact with other biomaterials, cells, and inorganic/organic materials. In this review article, six research topics on biointerfaces are described to show examples of state-of-art research approaches. First, biointerface design of nanoparticles for molecular detection is described. Functionalized gold nanoparticles can be used for sensitive detection of various target molecules, including chemical compounds and biomolecules, such as DNA, proteins, cells, and viruses. Second, the interaction between bacterial cell surfaces and material surfaces, including the introduction of advances in analytical methods and theoretical calculations, are explained as well as their applications to bioprocesses. Third, bioconjugation technologies for localizing functional proteins at biointerfaces are introduced, in particular, by focusing the potential of enzymes as a catalytic tool for designing different types of bioconjugates that function at biointerfaces. Forth topics is focusing on lipid-protein interaction in cell membranes as natural biointerfaces. Examples of membrane lipid engineering are introduced, and it is mentioned how their compositional profiles affect membrane protein functions. Fifth topic is the physical method for molecular delivery across the biointerface being developed currently, such as highly efficient nanoinjection, electroporation, and nanoneedle devices, in which the key is how to perforate the cell membrane. Final topic is the chemical design of lipid- or polymer-based RNA delivery carriers and their behavior on the cell interface, which are currently attracting attention as RNA vaccine technologies targeting COVID-19. Finally, future directions of biointerface studies are presented.

The Importance of Apparent pKa in the Development of Nanoparticles Encapsulating siRNA and mRNA.

Polymer and lipid nanoparticles have been extensively used as carriers to address the biological barriers encountered in siRNA and mRNA delivery. We summarize the crucial role of nanoparticle charge and ionizability in complexing RNAs, binding to biological components, escaping from the endosome, and releasing RNAs into the cytoplasm. We highlight the significant impact of the apparent pKa of nanoparticles on their efficacy and toxicity, and the importance of optimizing pKa in the development of lead formulations for RNAs. We also discuss the feasibility of fine-tuning the pKa in nanoparticles and the applications of this approach in the optimization of delivery systems for RNAs.

DNA-inspired nanomaterials for enhanced endosomal escape.

To realize RNA interference (RNAi) therapeutics, it is necessary to deliver therapeutic RNAs (such as small interfering RNA or siRNA) into cell cytoplasm. A major challenge of RNAi therapeutics is the endosomal entrapment of the delivered siRNA. In this study, we developed a family of delivery vehicles called Janus base nanopieces (NPs). They are rod-shaped nanoparticles formed by bundles of Janus base nanotubes (JBNTs) with RNA cargoes incorporated inside via charge interactions. JBNTs are formed by noncovalent interactions of small molecules consisting of a base component mimicking DNA bases and an amino acid side chain. NPs presented many advantages over conventional delivery materials. NPs efficiently entered cells via macropinocytosis similar to lipid nanoparticles while presenting much better endosomal escape ability than lipid nanoparticles; NPs escaped from endosomes via a "proton sponge" effect similar to cationic polymers while presenting significant lower cytotoxicity compared to polymers and lipids due to their noncovalent structures and DNA-mimicking chemistry. In a proof-of-concept experiment, we have shown that NPs are promising candidates for antiviral delivery applications, which may be used for conditions such as COVID-19 in the future.

Cytosolic delivery of nucleic acids: The case of ionizable lipid nanoparticles.

Ionizable lipid nanoparticles (LNPs) are the most clinically advanced nano-delivery system for therapeutic nucleic acids. The great effort put in the development of ionizable lipids with increased in vivo potency brought LNPs from the laboratory benches to the FDA approval of patisiran in 2018 and the ongoing clinical trials for mRNA-based vaccines against SARS-CoV-2. Despite these success stories, several challenges remain in RNA delivery, including what is known as "endosomal escape." Reaching the cytosol is mandatory for unleashing the therapeutic activity of RNA molecules, as their accumulation in other intracellular compartments would simply result in efficacy loss. In LNPs, the ability of ionizable lipids to form destabilizing non-bilayer structures at acidic pH is recognized as the key for endosomal escape and RNA cytosolic delivery. This is motivating a surge in studies aiming at designing novel ionizable lipids with improved biodegradation and safety profiles. In this work, we describe the journey of RNA-loaded LNPs across multiple intracellular barriers, from the extracellular space to the cytosol. In silico molecular dynamics modeling, in vitro high-resolution microscopy analyses, and in vivo imaging data are systematically reviewed to distill out the regulating mechanisms underlying the endosomal escape of RNA. Finally, a comparison with strategies employed by enveloped viruses to deliver their genetic material into cells is also presented. The combination of a multidisciplinary analytical toolkit for endosomal escape quantification and a nature-inspired design could foster the development of future LNPs with improved cytosolic delivery of nucleic acids.

Improved multidetector asymmetrical-flow field-flow fractionation method for particle sizing and concentration measurements of lipid-based nanocarriers for RNA delivery.

Lipid-based nanoparticles for RNA delivery (LNP-RNA) are revolutionizing the nanomedicine field, with one approved gene therapy formulation and two approved vaccines against COVID-19, as well as multiple ongoing clinical trials. As for other innovative nanopharmaceuticals (NPhs), the advancement of robust methods to assess their quality and safety profiles-in line with regulatory needs-is critical for facilitating their development and clinical translation. Asymmetric-flow field-flow fractionation coupled to multiple online optical detectors (MD-AF4) is considered a very versatile and robust approach for the physical characterisation of nanocarriers, and has been used successfully for measuring particle size, polydispersity and physical stability of lipid-based systems, including liposomes and solid lipid nanoparticles. However, the unique core structure of LNP-RNA, composed of ionizable lipids electrostatically complexed with RNA, and the relatively labile lipid-monolayer coating, is more prone to destabilization during focusing in MD-AF4 than previously characterised nanoparticles, resulting in particle aggregation and sample loss. Hence characterisation of LNP-RNA by MD-AF4 needs significant adaptation of the methods developed for liposomes. To improve the performance of MD-AF4 applied to LNP-RNA in a systematic and comprehensive manner, we have explored the use of the frit-inlet channel where, differently from the standard AF4 channel, the particles are relaxed hydrodynamically as they are injected. The absence of a focusing step minimizes contact between the particle and the membrane, reducing artefacts (e.g. sample loss, particle aggregation). Separation in a frit-inlet channel enables satisfactory reproducibility and acceptable sample recovery in the commercially available MD-AF4 instruments. In addition to slice-by-slice measurements of particle size, MD-AF4 also allows to determine particle concentration and the particle size distribution, demonstrating enhanced versatility beyond standard sizing measurements.