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Developing safe and effective delivery strategies for localizing messenger RNA (mRNA) payloads to the spleen is an important goal in the field of genetic medicine. Accomplishing this goal is challenging due to the instability, size, and charge of mRNA payloads. Here, we provide an analysis of non-viral delivery technologies that have been developed to deliver mRNA payloads to the spleen. Specifically, our review begins by outlining the unique anatomy and potential targets for mRNA delivery within the spleen. Next, we describe approaches in mRNA sequence engineering that can be used to improve mRNA delivery to the spleen. Then, we describe advances in non-viral carrier systems that can package and deliver mRNA payloads to the spleen, highlighting key advances in the literature in lipid nanoparticle (LNP) and polymer nanoparticle (PNP) technology platforms. Finally, we provide commentary and outlook on how splenic mRNA delivery may afford next-generation treatments for autoimmune disorders and cancers. In undertaking this approach, our goal with this review is to both establish a fundamental understanding of drug delivery challenges associated with localizing mRNA payloads to the spleen, while also broadly highlighting the potential to use these genetic medicines to treat disease.

Despite their successful implementation in the COVID-19 vaccines, lipid nanoparticles (LNPs) still face a central limitation in the delivery of mRNA payloads: endosomal trapping. Improving upon this inefficiency could afford improved drug delivery systems, paving the way toward safer and more effective mRNA-based medicines. Here, we present p olyphenolic n a nopa r ti c l e p l atforms (PARCELs) as effective mRNA delivery systems. In brief, our investigation begins with a computationally guided structural analysis of 1825 discrete polyphenolic structural data points across 73 diverse small molecule polyphenols and 25 molecular parameters. We then generate structurally diverse PARCELs, evaluating their in vitro mechanism and activity, ultimately highlighting the superior endosomal escape properties of PARCELs relative to analogous LNPs. Finally, we examine the in vivo biodistribution, protein expression, and therapeutic efficacy of PARCELs in mice. In undertaking this approach, the goal of this study is to establish PARCELs as viable delivery platforms for safe and effective mRNA delivery.

Neuroinflammation is a hallmark of neurodegenerative disorders including Alzheimer's disease (AD). Microglia, the brain's immune cells, express many of the AD-risk loci identified in genome wide association studies and present a promising target for anti-inflammatory RNA therapeutics but are difficult to transfect with current methods. Here, several lipid nanoparticle (LNP) formulations are examined, and a lead candidate that supports efficient RNA delivery in cultures of human stem cell-derived microglia-like cells (iMGLs) and animal models of neuroinflammation is identified. The lead microglia LNP (MG-LNP) formulation shows minimal toxicity and improves delivery efficiency to inflammatory iMGLs, suggesting a preference for delivery into activated microglia. Intraperitoneal injection of the MG-LNP formulation generates widespread expression of the delivered reporter construct in all organs, whereas local intracisternal injection directly into the cerebrospinal fluid leads to preferential expression in the brain. It is shown that LNP-mediated delivery of siRNA targeting the PU.1 transcription factor, a known AD-risk locus, successfully reduces PU.1 levels in iMGLs and reduces neuroinflammation in mice injected with LPS and in CK-p25 mice that mimic the chronic neuroinflammation seen in AD patients. The LNP formulation represents an effective RNA delivery vehicle when applied intrathecally and can be broadly utilized to test potential neuroinflammation-directed gene therapies., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

The concept of using mRNA to produce its own medicine in situ in the body makes it an ideal drug candidate, holding great potential to revolutionize the way we approach medicine. The unique characteristics of mRNA, as well as its customizable biomedical functions, call for the rational design of delivery systems to protect and transport mRNA molecules. In this review, a nanoparticle toolkit is presented for the development of mRNA-based therapeutics from a drug delivery perspective. Nano-delivery systems derived from either natural systems or chemical synthesis, in the nature of organic or inorganic materials, are summarised. Delivery strategies in controlling the tissue targeting and mRNA release, as well as the role of nanoparticles in building and boosting the activity of mRNA drugs, have also been introduced. In the end, our insights into the clinical and translational development of mRNA nano-drugs are presented., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Short interfering RNAs (siRNA) are a powerful class of genetic medicines whose clinical translation can be hindered by their suboptimal delivery properties in vivo . Here, we provide a clinically focused overview that summarizes ongoing siRNA clinical trials from the perspective of innovations in nonviral delivery strategies. More specifically, our review begins by highlighting the delivery barriers and physiochemical properties of siRNA that make it challenging to deliver it in vivo . We then provide commentary on specific delivery strategies, including sequence modification, siRNA ligand conjugation, and nanoparticle and exosomal packaging, each of which can be used to control the delivery of siRNA therapies in living systems. Last, we provide a summary table of ongoing siRNA clinical trials which also highlights the indication of use, target, and National Clinical Trial (NCT) number associated with each entry. In writing this review, our work aims to highlight the key challenges and strategies for effective nonviral siRNA delivery in vivo , while simultaneously summarizing information on ongoing clinical trials for siRNA therapy in humans.

Hypoxia is a common hallmark of human disease that is characterized by abnormally low oxygen levels in the body. While the effects of hypoxia on many small molecule-based drugs are known, its effects on several classes of next-generation medications including messenger RNA therapies warrant further study. Here, we provide an efficacy- and mechanism-driven study that details how hypoxia impacts the cellular response to mRNA therapies delivered using 4 different chemistries of lipid nanoparticles (LNPs, the frontrunner class of drug delivery vehicles for translational mRNA therapy utilized in the Moderna and Pfizer/BioNTech COVID-19 vaccines). Specifically, our work provides a comparative analysis as to how various states of oxygenation impact LNP-delivered mRNA expression, cellular association, endosomal escape, and intracellular ATP concentrations following treatment with 4 different LNPs across 3 different cell lines. In brief, we first identify that hypoxic cells express less LNP-delivered mRNA into protein than normoxic cells. Next, we identify generalizable cellular reoxygenation protocols that can reverse the negative effects that hypoxia imparts on LNP-delivered mRNA expression. Finally, mechanistic studies that utilize fluorescence-activated cell sorting, confocal microscopy, and enzyme inhibition reveal that decreases in mRNA expression correlate with decreases in intracellular ATP (rather than with differences in mRNA LNP uptake pathways). In presenting this data, we hope that our work provides a comprehensive efficacy and mechanism-driven study that explores the impact of differential oxygenation on LNP-delivered mRNA expression while simultaneously establishing fundamental criteria that may one day be useful for the development of mRNA drugs to treat hypoxia-associated disease.

Despite tremendous interest in gene therapies, the systemic delivery of nucleic acids still faces substantial challenges. To successfully administer nucleic acids, one approach is to encapsulate them in lipid nanoparticles (LNPs). However, LNPs administered intravenously substantially accumulate in the liver where they are taken up by the reticuloendothelial system (RES). Here, we administer prior to the LNPs a liposome designed to transiently occupy liver cells, the Nanoprimer. This study demonstrates that the pretreatment of mice with the Nanoprimer decreases the LNPs' uptake by the RES. By accumulating rapidly in the liver cells, the Nanoprimer improves the bioavailability of the LNPs encapsulating human erythropoietin (hEPO) mRNA or factor VII (FVII) siRNA, leading respectively to more hEPO production (by 32%) or FVII silencing (by 49%). The use of the Nanoprimer offers a new strategy to improve the systemic delivery of RNA-based therapeutics.

The ability to engineer immune function has transformed modern medicine, highlighted by the success of vaccinations and recent efforts in cancer immunotherapy. Further directions in programming the immune system focus on the design of immunomodulatory biomaterials that can recruit, engage with, and program immune cells locally in vivo. Here, we synthesized shear-thinning and self-healing polymer-nanoparticle (PNP) hydrogels as a tunable and injectable biomaterial platform for local dendritic cell (DC) recruitment. PNP gels were formed from two populations of poly(ethylene glycol)- block -polylactide (PEG- b -PLA) NPs with the same diameter but different PEG brush length (2 or 5 kDa). PEG- b -PLA NPs with the longer PEG brush exhibited improved gel formation following self-assembly and faster recovery after shear-thinning. In all cases, model protein therapeutics were released via Fickian diffusion in vitro, and minor differences in the release rate between the gel formulations were observed. PNP hydrogels were loaded with the DC cytokine CCL21 and injected subcutaneously in a murine model. CCL21-loaded PNP hydrogels recruited DCs preferentially to the site of injection in vivo relative to non-CCL21-loaded hydrogels. Thus, PNP hydrogels comprise a simple and tunable platform biomaterial for in vivo immunomodulation following minimally invasive subcutaneous injection.

mRNA therapeutics hold great potential for treating a variety of diseases through protein-replacement, immunomodulation, and gene editing. However, much like siRNA therapy the majority of progress in mRNA delivery has been confined to the liver. Previously, we demonstrated that poly(β-amino esters), a class of degradable polymers, are capable of systemic mRNA delivery to the lungs in mice when formulated into nanoparticles with poly(ethylene glycol)-lipid conjugates. Using experimental design, a statistical approach to optimization that reduces experimental burden, we demonstrate herein that these degradable polymer-lipid nanoparticles can be optimized in terms of polymer synthesis and nanoparticle formulation to achieve a multiple order-of-magnitude increase in potency. Furthermore, using genetically engineered Cre reporter mice, we demonstrate that mRNA is functionally delivered to both the lung endothelium and pulmonary immune cells, expanding the potential utility of these nanoparticles.

RNAs are a promising class of therapeutics given their ability to regulate protein concentrations at the cellular level. Developing safe and effective strategies to deliver RNAs remains important for realizing their full clinical potential. Here, we develop lipid nanoparticle formulations that can deliver short interfering RNAs (for gene silencing) or messenger RNAs (for gene upregulation). Specifically, we study how the tail length, tail geometry, and linker spacing in diketopiperazine lipid materials influences LNP potency with siRNAs and mRNAs. Eight lipid materials are synthesized, and 16 total formulations are screened for activity in vitro; the lead material is evaluated with mRNA for in vivo use and demonstrates luciferase protein expression in the spleen. In undertaking this approach, not only do we develop synthetic routes to delivery materials, but we also reveal structural criteria that could be useful for developing next-generation delivery materials for RNA therapeutics., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The induction of a strong cytotoxic T cell response is an important prerequisite for successful immunotherapy against many viral diseases and tumors. Nucleotide vaccines, including mRNA vaccines with their intracellular antigen synthesis, have been shown to be potent activators of a cytotoxic immune response. The intracellular delivery of mRNA vaccines to the cytosol of antigen presenting immune cells is still not sufficiently well understood. Here, we report on the development of a lipid nanoparticle formulation for the delivery of mRNA vaccines to induce a cytotoxic CD 8 T cell response. We show transfection of dendritic cells, macrophages, and neutrophils. The efficacy of the vaccine was tested in an aggressive B16F10 melanoma model. We found a strong CD 8 T cell activation after a single immunization. Treatment of B16F10 melanoma tumors with lipid nanoparticles containing mRNA coding for the tumor-associated antigens gp100 and TRP2 resulted in tumor shrinkage and extended the overall survival of the treated mice. The immune response can be further increased by the incorporation of the adjuvant LPS. In conclusion, the lipid nanoparticle formulation presented here is a promising vector for mRNA vaccine delivery, one that is capable of inducing a strong cytotoxic T cell response. Further optimization, including the incorporation of different adjuvants, will likely enhance the potency of the vaccine.

Therapeutic nucleic acids hold great promise for the treatment of disease but require vectors for safe and effective delivery. Synthetic nanoparticle vectors composed of poly(β-amino esters) (PBAEs) and nucleic acids have previously demonstrated potential utility for local delivery applications. To expand this potential utility to include systemic delivery of mRNA, hybrid polymer-lipid nanoformulations for systemic delivery to the lungs were developed. Through coformulation of PBAEs with lipid-polyethylene glycol (PEG), mRNA formulations were developed with increased serum stability and increased in vitro potency. The formulations were capable of functional delivery of mRNA to the lungs after intravenous administration in mice. To our knowledge, this is the first report of the systemic administration of mRNA for delivery to the lungs using degradable polymer-lipid nanoparticles., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Thousands of human diseases could be treated by selectively controlling the expression of specific proteins in vivo. A new series of alkenyl amino alcohol (AAA) ionizable lipid nanoparticles (LNPs) capable of delivering human mRNA with unprecedented levels of in vivo efficacy is demonstrated. This study highlights the importance of utilizing synthesis tools in tandem with biological inspiration to understand and improve nucleic acid delivery in vivo., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intracellular delivery of messenger RNA (mRNA) has the potential to induce protein production for many therapeutic applications. Although lipid nanoparticles have shown considerable promise for the delivery of small interfering RNAs (siRNA), their utility as agents for mRNA delivery has only recently been investigated. The most common siRNA formulations contain four components: an amine-containing lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency. Here, we develop a generalized strategy to optimize lipid nanoparticle formulations for mRNA delivery to the liver in vivo using Design of Experiment (DOE) methodologies including Definitive Screening and Fractional Factorial Designs. By simultaneously varying lipid ratios and structures, we developed an optimized formulation which increased the potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles 7-fold relative to formulations previously used for siRNA delivery. Key features of this optimized formulation were the incorporation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and increased ionizable lipid:mRNA weight ratios. Interestingly, the optimized lipid nanoparticle formulation did not improve siRNA delivery, indicating differences in optimized formulation parameter design spaces for siRNA and mRNA. We believe the general method described here can accelerate in vivo screening and optimization of nanoparticle formulations with large multidimensional design spaces.

One of the most significant challenges in the development of clinically viable delivery systems for RNA interference therapeutics is to understand how molecular structures influence delivery efficacy. Here, we have synthesized 1,400 degradable lipidoids and evaluate their transfection ability and structure-function activity. We show that lipidoid nanoparticles mediate potent gene knockdown in hepatocytes and immune cell populations on IV administration to mice (siRNA EC50 values as low as 0.01 mg kg(-1)). We identify four necessary and sufficient structural and pKa criteria that robustly predict the ability of nanoparticles to mediate greater than 95% protein silencing in vivo. Because these efficacy criteria can be dictated through chemical design, this discovery could eliminate our dependence on time-consuming and expensive cell culture assays and animal testing. Herein, we identify promising degradable lipidoids and describe new design criteria that reliably predict in vivo siRNA delivery efficacy without any prior biological testing.

Therapeutic nucleic acids hold great promise for the treatment of disease but require vectors for safe and effective delivery. Synthetic nanoparticle vectors composed of poly(β-amino esters) (PBAEs) and nucleic acids have previously demonstrated potential utility for local delivery applications. To expand this potential utility to include systemic delivery of mRNA, hybrid polymer-lipid nanoformulations for systemic delivery to the lungs were developed. Through coformulation of PBAEs with lipid-polyethylene glycol (PEG), mRNA formulations were developed with increased serum stability and increased in vitro potency. The formulations were capable of functional delivery of mRNA to the lungs after intravenous administration in mice. To our knowledge, this is the first report of the systemic administration of mRNA for delivery to the lungs using degradable polymer-lipid nanoparticles. [ABSTRACT FROM AUTHOR]

Influenza outbreaks are a major burden worldwide annually. While seasonal vaccines do provide protection against infection, they are limited in that they need to be updated every year to account for the constantly mutating virus. Recently, lipid nanoparticles (LNPs) encapsulating mRNA have seen major success as a vaccine platform for SARS-CoV-2. Herein, we applied LNPs to deliver an mRNA encoding a computationally optimized broadly active (COBRA) influenza immunogen. These COBRA mRNA LNPs induced a broadly active neutralizing antibody response and protection after lethal influenza challenge. To further increase the immunogenicity of the COBRA mRNA LNPs, we combined them with acetalated dextran microparticles encapsulating a STING agonist. Contrary to recent findings, the STING agonist decreased the immunogenicity of the COBRA mRNA LNPs which was likely due to a decrease in mRNA translation as shown in vitro. Overall, this work aids in future selection of adjuvants to use with mRNA LNP vaccines. [Display omitted] [ABSTRACT FROM AUTHOR]