RNA-based Therapeutics Research Articles

Multimeric RNAs for efficient RNA-based therapeutics and vaccines.

There has been a growing interest in RNA therapeutics globally, and much progress has been made in this area, which has been further accelerated by the clinical applications of RNA-based vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Following these successful clinical trials, various technologies have been developed to improve the efficacy of RNA-based drugs. Multimerization of RNA therapeutics is one of the most attractive approaches to ensure high stability, high efficacy, and prolonged action of RNA-based drugs. In this review, we offer an overview of the representative approaches for generating repetitive functional RNAs by chemical conjugation, structural self-assembly, enzymatic elongation, and self-amplification. The therapeutic and vaccine applications of engineered multimeric RNAs in various diseases have also been summarized. By outlining the current status of multimeric RNAs, the potential of multimeric RNA as a promising treatment strategy is highlighted.

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured “superfolder” mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

The Fra-1/AP-1 Oncoprotein: From the “Undruggable” Transcription Factor to Therapeutic Targeting

Simple SummaryCancer-associated mortality largely depends on metastatic dissemination. Metastasizing cancer cells exhibit drastic phenotypic changes, including the ability to migrate, invade surrounding tissues, survive in the bloodstream, adapt to different microenvironments, and resist to therapeutic treatments. These changes depend on the genetic reprogramming orchestrated by relatively few players (transcription factors). Among the components of the dimeric transcription factor AP-1, the nuclear oncoprotein Fra-1 is strongly implicated in metastasis mechanisms. Therefore, Fra-1, along with other proinvasive transcription factors, represents an ideal therapeutic target. However, as for other DNA-binding proteins, the design of inhibitory drugs is hampered by the structural features of Fra-1. In this Review, after summarizing the Fra-1 functions and mechanisms of accumulation in invasive tumors, we survey the possible application of multiple strategies and emerging technologies aimed at the inhibition of Fra-1 expression and activity, to prevent metastatic dissemination and therapeutic resistance. The genetic and epigenetic changes affecting transcription factors, coactivators, and chromatin modifiers are key determinants of the hallmarks of cancer. The acquired dependence on oncogenic transcriptional regulators, representing a major determinant of cancer cell vulnerability, points to transcription factors as ideal therapeutic targets. However, given the unavailability of catalytic activities or binding pockets for small-molecule inhibitors, transcription factors are generally regarded as undruggable proteins. Among components of the AP-1 complex, the FOS-family transcription factor Fra-1, encoded by FOSL1, has emerged as a prominent therapeutic target. Fra-1 is overexpressed in most solid tumors, in response to the BRAF-MAPK, Wnt-beta-catenin, Hippo-YAP, IL-6-Stat3, and other major oncogenic pathways. In vitro functional analyses, validated in onco-mouse models and corroborated by prognostic correlations, show that Fra-1-containing dimers control tumor growth and disease progression. Fra-1 participates in key mechanisms of cancer cell invasion, Epithelial-to-Mesenchymal Transition, and metastatic spreading, by driving the expression of EMT-inducing transcription factors, cytokines, and microRNAs. Here we survey various strategies aimed at inhibiting tumor growth, metastatic dissemination, and drug resistance by interfering with Fra-1 expression, stability, and transcriptional activity. We summarize several tools aimed at the design and tumor-specific delivery of Fra-1/AP-1-specific drugs. Along with RNA-based therapeutics targeting the FOSL1 gene, its mRNA, or cognate regulatory circRNAs, we will examine the exploitation of blocking peptides, small molecule inhibitors, and innovative Fra-1 protein degraders. We also consider the possible caveats concerning Fra-1 inhibition in specific therapeutic contexts. Finally, we discuss a recent suicide gene therapy-based approach, aimed at selectively killing the Fra-1-overexpressing neoplastic cells.

Secondary structural ensembles of the SARS-CoV-2 RNA genome in infected cells

SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Although population average structure models of the genome were recently reported, there is little experimental data on native structural ensembles, and most structures lack functional characterization. Here we report secondary structure heterogeneity of the entire SARS-CoV-2 genome in two lines of infected cells at single nucleotide resolution. Our results reveal alternative RNA conformations across the genome and at the critical frameshifting stimulation element (FSE) that are drastically different from prevailing population average models. Importantly, we find that this structural ensemble promotes frameshifting rates much higher than the canonical minimal FSE and similar to ribosome profiling studies. Our results highlight the value of studying RNA in its full length and cellular context. The genomic structures detailed here lay groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.

RNA-based Therapeutics in Cardiology: Dawn of a New Era?

RNA-based Therapeutics in Cardiology: Dawn of a New Era?

Ultraviolet Photodissociation and Activated Electron Photodetachment Mass Spectrometry for Top-Down Sequencing of Modified Oligoribonucleotides.

With the increased development of new RNA-based therapeutics, the need for robust analytical methods for confirming sequences and mapping modifications has accelerated. Characterizing modified ribonucleic acids using mass spectrometry is challenging because diagnostic fragmentation may be suppressed for modified nucleotides, thus hampering complete sequence coverage and the confident localization of modifications. Ultraviolet photodissociation (UVPD) has shown great potential for the characterization of nucleic acids due to extensive backbone fragmentation. Activated electron photodetachment dissociation (a-EPD) has also been used as an alternative to capitalize on the dominant charge-reduction pathway prevalent in UVPD, facilitate dissociation, and produce high abundances of fragment ions. Here, we compare higher-energy collisional activation (HCD), UVPD using 193 and 213 nm photons, and a-EPD for the top-down sequencing of modified nucleic acids, including methylated, phosphorothioate, and locked nucleic acid-modified DNA. The presence of these modifications alters the fragmentation pathways observed upon UVPD and a-EPD, and extensive backbone cleavage is observed that results in the production of fragment ions that retain the modifications and allow them to be pinpointed. LNA and 2'-O-methoxy phosphorothioate modifications caused a significant suppression of fragmentation for UVPD but not for a-EPD, whereas phosphorothioate bonds did not cause any significant suppression for either method. The incorporation of 2'-O-methyl modifications suppressed fragmentation of the antisense strand of patisiran, which resulted in some gaps in sequence coverage. However, UVPD provided the highest sequence coverage when compared to a-EPD.

Nanoparticle-based delivery strategies of multifaceted immunomodulatory RNA for cancer immunotherapy

Although cancer immunotherapy has emerged as a novel cancer treatment modality, it still suffers from low therapeutic efficacy in clinics due to the presence of a low number of activated immune cells and immunosuppressive factors in the tumor microenvironment (TME). Immunomodulatory ribonucleic acids (RNAs) have been developed to improve the therapeutic efficacy of cancer immunotherapy through either regulating target cell functions [i.e., messenger RNA (mRNA) or small interfering RNA (siRNA)] or stimulating immune cells [i.e., toll-like receptors (TLRs) or cytosolic retinoic acid-inducible gene I (RIG-I) agonist]. However, RNA-based therapeutics face many biological barriers, including ineffective delivery to target cells, degradation by ribonucleases (RNases), and difficulties in passing through the cellular membranes. In this review, we discuss nanoparticle-based delivery strategies that can overcome these hurdles to enhance RNA-based immunomodulation in cancer immunotherapy. Various nanoparticle-based delivery has been reported to increase the delivery efficacy of RNAs, by improving cellular uptake, RNA stability, and accumulation at the desired sites (target cells and intracellular compartments). The nanoparticle-based delivery of multifaceted immunomodulatory RNAs could enhance cancer immunotherapy through the regulating functions of immune cells, tumor cells, and immunosuppressive factors as well as stimulating the immune cells by recognition of endosomal TLRs and cytosolic RIG-I. Nanotechnology-assisted RNA-based therapeutics are expected to offer tremendous potential and advances for treating cancer, viral infections, and other diseases.

MiR-182 targeting reprograms tumor-associated macrophages and limits breast cancer progression.

The protumor roles of alternatively activated (M2) tumor-associated macrophages (TAMs) have been well established, and macrophage reprogramming is an important therapeutic goal. However, the mechanisms of TAM polarization remain incompletely understood, and effective strategies for macrophage targeting are lacking. Here, we show that miR-182 in macrophages mediates tumor-induced M2 polarization and can be targeted for therapeutic macrophage reprogramming. Constitutive miR-182 knockout in host mice and conditional knockout in macrophages impair M2-like TAMs and breast tumor development. Targeted depletion of macrophages in mice blocks the effect of miR-182 deficiency in tumor progression while reconstitution of miR-182-expressing macrophages promotes tumor growth. Mechanistically, cancer cells induce miR-182 expression in macrophages by TGFβ signaling, and miR-182 directly suppresses TLR4, leading to NFκb inactivation and M2 polarization of TAMs. Importantly, therapeutic delivery of antagomiR-182 with cationized mannan-modified extracellular vesicles effectively targets macrophages, leading to miR-182 inhibition, macrophage reprogramming, and tumor suppression in multiple breast cancer models of mice. Overall, our findings reveal a crucial TGFβ/miR-182/TLR4 axis for TAM polarization and provide rationale for RNA-based therapeutics of TAM targeting in cancer.

Advanced drug delivery systems for enhancing the efficacy of RNA-based therapeutics

Background: RNA-based therapeutics, including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and messenger RNAs (mRNAs), offer significant promise in treating genetic and acquired diseases by targeting specific RNA sequences, encoding therapeutic proteins, or facilitating genome editing. However, the effective delivery of these RNA therapeutics remains a major challenge due to their large size, negative charge, and susceptibility to degradation. Aim: This review aims to explore advanced drug delivery systems developed to enhance the efficacy of RNA-based therapeutics, focusing on both viral and non-viral methods, and to evaluate the progress and limitations of these systems in clinical applications. Methods: The review synthesizes recent advancements in RNA delivery technologies, including viral vectors, lipid nanoparticles (LNPs), polymer-based nanoparticles, and hybrid systems. It also examines various targeting strategies such as passive and active targeting to improve the specificity and efficiency of RNA delivery. Results: Significant progress has been made with both viral and non-viral delivery systems. Viral vectors, though effective, face challenges related to immunogenicity and production costs. Non-viral systems, particularly lipid nanoparticles and polymer-based carriers, have shown promising results, with several FDA-approved products demonstrating clinical efficacy. Advances in targeting strategies, including ligand-based and antibody-based methods, have improved the precision of RNA delivery.

Mining LTR-retrotransposon genes for mRNA delivery

RNA-based therapeutics commonly use exogenous components to shuttle their cargo, leading to nonselectivity or immunogenicity. Segel et al. have elucidated an endogenous modular retroviral-like delivery system capable of encapsulating mRNA, which elicits effective transport inside cells, priming the development of endogenous vectors for gene delivery for amelioration of disease.

Delivery strategies of RNA therapeutics to leukocytes

Harnessing RNA-based therapeutics for cancer, inflammation, and viral diseases is hindered by poor delivery of therapeutic RNA molecules. Targeting leukocytes to treat these conditions holds great promise, as they are key participants in their initiation, drug response, and treatment. The various extra- and intra-cellular obstacles that impediment the clinical implementation of therapeutic RNA can be overcome by utilizing drug delivery systems. However, delivery of therapeutic RNA to leukocytes poses an even greater challenge as these cells are difficult to reach and transfect upon systemic administration. This review briefly describes the existing successful delivery strategies that efficiently target leukocytes in vivo and discuss their potential clinical applicability.

Recent Advances in RNA Therapy and Its Carriers to Treat the Single-Gene Neurological Disorders.

The development of new sequencing technologies in the post-genomic era has accelerated the identification of causative mutations of several single gene disorders. Advances in cell and animal models provide insights into the underlining pathogenesis, which facilitates the development and maturation of new treatment strategies. The progress in biochemistry and molecular biology has established a new class of therapeutics—the short RNAs and expressible long RNAs. The sequences of therapeutic RNAs can be optimized to enhance their stability and translatability with reduced immunogenicity. The chemically-modified RNAs can also increase their stability during intracellular trafficking. In addition, the development of safe and high efficiency carriers that preserves the integrity of therapeutic RNA molecules also accelerates the transition of RNA therapeutics into the clinic. For example, for diseases that are caused by genetic defects in a specific protein, an effective approach termed “protein replacement therapy” can provide treatment through the delivery of modified translatable mRNAs. Short interference RNAs can also be used to treat diseases caused by gain of function mutations or restore the splicing aberration defects. Here we review the applications of newly developed RNA-based therapeutics and its delivery and discuss the clinical evidence supporting the potential of RNA-based therapy in single-gene neurological disorders.

Gene Therapy Using Nanocarriers for Pancreatic Ductal Adenocarcinoma: Applications and Challenges in Cancer Therapeutics.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide, and its incidence is increasing. PDAC often shows resistance to several therapeutic modalities and a higher recurrence rate after surgical treatment in the early localized stage. Combination chemotherapy in advanced pancreatic cancer has minimal impact on overall survival. RNA interference (RNAi) is a promising tool for regulating target genes to achieve sequence-specific gene silencing. Here, we summarize RNAi-based therapeutics using nanomedicine-based delivery systems that are currently being tested in clinical trials and are being developed for the treatment of PDAC. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing has been widely used for the development of cancer models as a genetic screening tool for the identification and validation of therapeutic targets, as well as for potential cancer therapeutics. This review discusses current advances in CRISPR/Cas9 technology and its application to PDAC research. Continued progress in understanding the PDAC tumor microenvironment and nanomedicine-based gene therapy will improve the clinical outcomes of patients with PDAC.

Brain delivering RNA-based therapeutic strategies by targeting mTOR pathway for axon regeneration after central nervous system injury.

Injuries to the central nervous system (CNS) such as stroke, brain, and spinal cord trauma often result in permanent disabilities because adult CNS neurons only exhibit limited axon regeneration. The brain has a surprising intrinsic capability of recovering itself after injury. However, the hostile extrinsic microenvironment significantly hinders axon regeneration. Recent advances have indicated that the inactivation of intrinsic regenerative pathways plays a pivotal role in the failure of most adult CNS neuronal regeneration. Particularly, substantial evidence has convincingly demonstrated that the mechanistic target of rapamycin (mTOR) signaling is one of the most crucial intrinsic regenerative pathways that drive axonal regeneration and sprouting in various CNS injuries. In this review, we will discuss the recent findings and highlight the critical roles of mTOR pathway in axon regeneration in different types of CNS injury. Importantly, we will demonstrate that the reactivation of this regenerative pathway can be achieved by blocking the key mTOR signaling components such as phosphatase and tensin homolog (PTEN). Given that multiple mTOR signaling components are endogenous inhibitory factors of this pathway, we will discuss the promising potential of RNA-based therapeutics which are particularly suitable for this purpose, and the fact that they have attracted substantial attention recently after the success of coronavirus disease 2019 vaccination. To specifically tackle the blood-brain barrier issue, we will review the current technology to deliver these RNA therapeutics into the brain with a focus on nanoparticle technology. We will propose the clinical application of these RNA-mediated therapies in combination with the brain-targeted drug delivery approach against mTOR signaling components as an effective and feasible therapeutic strategy aiming to enhance axonal regeneration for functional recovery after CNS injury.

Aptamer-based RNA-bio-drugs for the combined therapy of GBM

This paper describes a novel combined approach to targeting GBM with multiple RNA-based therapeutics. Overall, the results demonstrate the good potential of a smart safe and clinically applicable combined regimen for the treatment of glioblastoma, opening up a new avenue in the treatment of this aggressive disease

TREAT: Therapeutic RNAs exploration inspired by artificial intelligence technology

Recent advances in RNA engineering have enabled the development of RNA-based therapeutics for a broad spectrum of applications. Developing RNA therapeutics start with targeted RNA screening and move to the drug design and optimization. However, existing target screening tools ignore noncoding RNAs and their disease-relevant regulatory relationships. And designing therapeutic RNAs encounters high computational complexity of multi-objective optimization to overcome the immunogenicity, instability and inefficient translational production. To unlock the therapeutic potential of noncoding RNAs and enable one-stop screening and design of therapeutic RNAs, we have built the platform TREAT. It incorporates 43,087,953 regulatory relationships between coding and noncoding genes from 81 biological networks under different physiological conditions. TREAT introduces graph representation learning with Random Walk Diffusions to perform disease-relevant target screening, in addition to the commonly utilized Topological Degree and PageRank algorithms. Design and optimization of large RNAs or interfering RNAs are both available. To reduce the computational complexity of multi-objective optimization for large RNA, we stratified the features into local and global features. The local features are evaluated on the fixed-length or dynamic-length local bins, whereas the latter are inspired by AI language models of protein sequence. Then the global assessment is performed on refined candidates, thus reducing the enormous search space. Overall, TREAT is a one-stop platform for the screening and designing of therapeutic RNAs, with particular attention to noncoding RNAs and cutting-edge AI technology embedded, leading the progress of innovativetherapeutics for challenging diseases. TREAT is freely accessible at https://rna.org.cn/treat.

Polymers Strive for Accuracy: From Sequence-Defined Polymers to mRNA Vaccines against COVID-19 and Polymers in Nucleic Acid Therapeutics.

Unquestionably, polymers have influenced the world over the past 100 years. They are now more crucial than ever since the COVID-19 pandemic outbreak. The pandemic paved the way for certain polymers to be in the spotlight, namely sequence-defined polymers such as messenger ribonucleic acid (mRNA), which was the first type of vaccine to be authorized in the U.S. and Europe to protect against the SARS-CoV-2 virus. This rise of mRNA will probably influence scientific research concerning nucleic acids in general and RNA therapeutics in specific. In this Perspective, we highlight the recent trends in sequence-controlled and sequence-defined polymers. Then we discuss mRNA vaccines as an example to illustrate the need of ultimate sequence control to achieve complex functions such as specific activation of the immune system. We briefly present how mRNA vaccines are produced, the importance of modified nucleotides, the characteristic features, and the advantages and challenges associated with this class of vaccines. Finally, we discuss the chances and opportunities for polymer chemistry to provide solutions and contribute to the future progress of RNA-based therapeutics. We highlight two particular roles of polymers in this context. One represents conjugation of polymers to nucleic acids to form biohybrids. The other is concerned with advanced polymer-based carrier systems for nucleic acids. We believe that polymers can help to address present problems of RNA-based therapeutic technologies and impact the field beyond the COVID-19 pandemic.

CyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2ʹ,3ʹ cyclic phosphates and 5ʹ hydroxyl ends

ABSTRACT Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5′-fragment with a 2′,3′-cyclic phosphate and a 3′-fragment with a 5′-hydroxyl. They are widely applied for the construction of synthetic RNA devices and RNA-based therapeutics. However, the targeted discovery of self-cleaving ribozymes remains a major challenge. We developed a transcriptome-wide method, called cyPhyRNA-seq, to screen for ribozyme cleavage fragments in total RNA extract. This approach employs the specific ligation-based capture of ribozyme 5′-fragments using a variant of the Arabidopsis thaliana tRNA ligase we engineered. To capture ribozyme 3′-fragments, they are enriched from total RNA by enzymatic treatments. We optimized and enhanced the individual steps of cyPhyRNA-seq in vitro and in spike-in experiments. Then, we applied cyPhyRNA-seq to total RNA isolated from the bacterium Desulfovibrio vulgaris and detected self-cleavage of the three predicted type II hammerhead ribozymes, whose activity had not been examined to date. cyPhyRNA-seq can be used for the global analysis of active self-cleaving ribozymes with the advantage to capture both ribozyme cleavage fragments from total RNA. Especially in organisms harbouring many self-cleaving RNAs, cyPhyRNA-seq facilitates the investigation of cleavage activity. Moreover, this method has the potential to be used to discover novel self-cleaving ribozymes in different organisms.

An RNAi therapeutic targeting hepatic DGAT2 in a genetically obese mouse model of nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplant, with no approved therapeutics available. Although the exact molecular mechanism of NASH progression is not well understood, a widely held hypothesis is that fat accumulation is the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long-term gene silencing with single subcutaneous administration. Here, we identified a hyper-functional, fully chemically stabilized GalNAc-conjugated small interfering RNA (siRNA) targeting DGAT2 (Dgat2-1473) that, upon injection, elicits up to 3months of DGAT2 silencing (>80%-90%, p< 0.0001) in wild-type and NSG-PiZ "humanized" mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (>85%, p< 0.0001) without increased accumulation of diglycerides, resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat did not translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH.

Targeting the Inside of Cells with Biologicals: Chemicals as a Delivery Strategy.

Delivering macromolecules into the cytosol or nucleus is possible in vitro for DNA, RNA and proteins, but translation for clinical use has been limited. Therapeutic delivery of macromolecules into cells requires overcoming substantially higher barriers compared to the use of small molecule drugs or proteins in the extracellular space. Breakthroughs like DNA delivery for approved gene therapies and RNA delivery for silencing of genes (patisiran, ONPATTRO®, Alnylam Pharmaceuticals, Cambridge, MA, USA) or for vaccination such as the RNA-based coronavirus disease 2019 (COVID-19) vaccines demonstrated the feasibility of using macromolecules inside cells for therapy. Chemical carriers are part of the reason why these novel RNA-based therapeutics possess sufficient efficacy for their clinical application. A clear advantage of synthetic chemicals as carriers for macromolecule delivery is their favourable properties with respect to production and storage compared to more bioinspired vehicles like viral vectors or more complex drugs like cellular therapies. If biologicals can be applied to intracellular targets, the druggable space is substantially broadened by circumventing the limited utility of small molecules for blocking protein–protein interactions and the limitation of protein-based drugs to the extracellular space. An in depth understanding of the macromolecular cargo types, carrier types and the cell biology of delivery is crucial for optimal application and further development of biologicals inside cells. Basic mechanistic principles of the molecular and cell biological aspects of cytosolic/nuclear delivery of macromolecules, with particular consideration of protein delivery, are reviewed here. The efficiency of macromolecule delivery and applications in research and therapy are highlighted.