Messenger RNA (mRNA) has emerged as a new category of therapeutic agent to prevent and treat various diseases. To function in vivo, mRNA requires safe, effective and stable delivery systems that protect the nucleic acid from degradation and that allow cellular uptake and mRNA release. Lipid nanoparticles have successfully entered the clinic for the delivery of mRNA; in particular, lipid nanoparticle-mRNA vaccines are now in clinical use against coronavirus disease 2019 (COVID-19), which marks a milestone for mRNA therapeutics. In this Review, we discuss the design of lipid nanoparticles for mRNA delivery and examine physiological barriers and possible administration routes for lipid nanoparticle-mRNA systems. We then consider key points for the clinical translation of lipid nanoparticle-mRNA formulations, including good manufacturing practice, stability, storage and safety, and highlight preclinical and clinical studies of lipid nanoparticle-mRNA therapeutics for infectious diseases, cancer and genetic disorders. Finally, we give an outlook to future possibilities and remaining challenges for this promising technology.

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Lipid nanoparticles for mRNA delivery.

Treating patients who have cancer with vaccines that stimulate a targeted immune response is conceptually appealing, but cancer vaccine trials have not been successful in late-stage patients with treatment-refractory tumours1,2. We are testing melanoma FixVac (BNT111)—an intravenously administered liposomal RNA (RNA-LPX) vaccine, which targets four non-mutated, tumour-associated antigens that are prevalent in melanoma—in an ongoing, first-in-human, dose-escalation phase I trial in patients with advanced melanoma (Lipo-MERIT trial, ClinicalTrials.gov identifier NCT02410733). We report here data from an exploratory interim analysis that show that melanoma FixVac, alone or in combination with blockade of the checkpoint inhibitor PD1, mediates durable objective responses in checkpoint-inhibitor (CPI)-experienced patients with unresectable melanoma. Clinical responses are accompanied by the induction of strong CD4+ and CD8+ T cell immunity against the vaccine antigens. The antigen-specific cytotoxic T-cell responses in some responders reach magnitudes typically reported for adoptive T-cell therapy, and are durable. Our findings indicate that RNA-LPX vaccination is a potent immunotherapy in patients with CPI-experienced melanoma, and suggest the general utility of non-mutant shared tumour antigens as targets for cancer vaccination. Results of an exploratory interim analysis from a phase I trial show that an RNA vaccine targeted towards four melanoma-associated antigens produces durable objective responses in patients with melanoma that are accompanied by strong CD4+ and CD8+ T-cell immunity.

https://www.nature.com/articles/s41586-020-2537-9.pdf?proof=t

An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma.

Nucleic acid separation is an increasingly important tool for molecular biology. Before modern technologies could be used, nucleic acid separation had been a time- and work-consuming process based on several extraction and centrifugation steps, often limited by small yields and low purities of the separation products, and not suited for automation and up-scaling. During the last few years, specifically functionalised magnetic particles were developed. Together with an appropriate buffer system, they allow for the quick and efficient purification directly after their extraction from crude cell extracts. Centrifugation steps were avoided. In addition, the new approach provided for an easy automation of the entire process and the isolation of nucleic acids from larger sample volumes. This review describes traditional methods and methods based on magnetic particles for nucleic acid purification. The synthesis of a variety of magnetic particles is presented in more detail. Various suppliers of magnetic particles for nucleic acid separation as well as suppliers offering particle-based kits for a variety of different sample materials are listed. Furthermore, commercially available manual magnetic separators and automated systems for magnetic particle handling and liquid handling are mentioned.

Magnetic particles for separation and purification of nucleic acids

RNA-based gene therapy requires therapeutic RNA to function inside target cells without eliciting unwanted immune responses. RNA can be ferried into cells using non-viral drug delivery systems, which circumvent the limitations of viral delivery vectors. Here, we review the growing number of RNA therapeutic classes, their molecular mechanisms of action, and the design considerations for their respective delivery platforms. We describe polymer-based, lipid-based, and conjugate-based drug delivery systems, differentiating between those that passively and those that actively target specific cell types. Finally, we describe the path from preclinical drug delivery research to clinical approval, highlighting opportunities to improve the efficiency with which new drug delivery systems are discovered. RNA therapies can be used to manipulate gene expression or produce therapeutic proteins. Here, the authors describe the growing number of RNA therapies and their molecular mechanisms of action. They also discuss the path from preclinical drug delivery research to clinical approval of these drugs. 

/pdf/drug-delivery-systems-for-rna-therapeutics-2dmuidva.pdf

Drug delivery systems for RNA therapeutics

The challenge and prospect of mRNA therapeutics landscape.

Chimeric antigen receptor (CAR)-T cells have shown efficacy in patients with B cell malignancies. Yet, their application for solid tumors has challenges that include limited cancer-specific targets and nonpersistence of adoptively transferred CAR-T cells. Here, we introduce the developmentally regulated tight junction protein claudin 6 (CLDN6) as a CAR target in solid tumors and a strategy to overcome inefficient CAR-T cell stimulation in vivo. We demonstrate that a nanoparticulate RNA vaccine, designed for body-wide delivery of the CAR antigen into lymphoid compartments, stimulates adoptively transferred CAR-T cells. Presentation of the natively folded target on resident antigen-presenting cells promotes cognate and selective expansion of CAR-T cells. Improved engraftment of CAR-T cells and regression of large tumors in difficult-to-treat mouse models was achieved at subtherapeutic CAR-T cell doses.

https://science.sciencemag.org/content/sci/early/2019/12/30/science.aay5967.full.pdf

An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors

Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.

Monitoring Translation Activity of mRNA-Loaded Nanoparticles in Mice

Chimeric antigen receptor (CAR) T cells are efficacious in patients with B-cell malignancies, while their activity is limited in patients with solid tumors. We developed a novel heterodimeric TCR-like CAR (TCAR) designed to achieve optimal chain pairing and integration into the T-cell CD3 signaling complex. The TCAR mediated high antigen sensitivity and potent antigen-specific T-cell effector functions in short-term in vitro assays. Both persistence and functionality of TCAR T cells were augmented by provision of costimulatory signals, which improved proliferation in vitro and in vivo. Combination with a nanoparticulate RNA vaccine, developed for in vivo expansion of CAR T cells, promoted tightly controlled expansion, survival, and antitumor efficacy of TCAR T cells in vivo. Significance: A novel TCAR is tightly controlled by RNA vaccine–mediated costimulation and may provide an alternative to second-generation CARs for the treatment of solid tumors.

https://aacrjournals.org/cancerrescommun/article-pdf/2/8/827/3192047/crc-21-0154.pdf

Nina Hayduk

Papers

An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors

Monitoring Translation Activity of mRNA-Loaded Nanoparticles in Mice

A TCR-like CAR Promotes Sensitive Antigen Recognition and Controlled T-cell Expansion Upon mRNA Vaccination