Therapeutic application of circular RNA aptamers in a mouse model of psoriasis.

Efforts to advance RNA aptamers as a new therapeutic modality have been limited by their susceptibility to degradation and immunogenicity. In a previous study, we demonstrated synthesized short double-stranded region-containing circular RNAs (ds-cRNAs) with minimal immunogenicity targeted to dsRNA-activated protein kinase R (PKR). Here we test the therapeutic potential of ds-cRNAs in a mouse model of imiquimod-induced psoriasis. We find that genetic supplementation of ds-cRNAs leads to inhibition of PKR, resulting in alleviation of downstream interferon-α and dsRNA signals and attenuation of psoriasis phenotypes. Delivery of ds-cRNAs by lipid nanoparticles to the spleen attenuates PKR activity in examined splenocytes, resulting in reduced epidermal thickness. These findings suggest that ds-cRNAs represent a promising approach to mitigate excessive PKR activation for therapeutic purposes.

Endosomal escape: A bottleneck for LNP-mediated therapeutics.

Lipid nanoparticles (LNPs) have recently emerged as a powerful and versatile clinically approved platform for nucleic acid delivery, specifically for mRNA vaccines. A major bottleneck in the field is the release of mRNA-LNPs from the endosomal pathways into the cytosol of cells where they can execute their encoded functions. The data regarding the mechanism of these endosomal escape processes are limited and contradicting. Despite extensive research, there is no consensus regarding the compartment of escape, the cause of the inefficient escape and are currently lacking a robust method to detect the escape. Here, we review the currently known mechanisms of endosomal escape and the available methods to study this process. We critically discuss the limitations and challenges of these methods and the possibilities to overcome these challenges. We propose that the development of currently lacking robust, quantitative high-throughput techniques to study endosomal escape is timely and essential. A better understanding of this process will enable better RNA-LNP designs with improved efficiency to unlock new therapeutic modalities.

Targeting cancer with mRNA-lipid nanoparticles: key considerations and future prospects.

Harnessing mRNA-lipid nanoparticles (LNPs) to treat patients with cancer has been an ongoing research area that started before these versatile nanoparticles were successfully used as COVID-19 vaccines. Currently, efforts are underway to harness this platform for oncology therapeutics, mainly focusing on cancer vaccines targeting multiple neoantigens or direct intratumoural injections of mRNA-LNPs encoding pro-inflammatory cytokines. In this Review, we describe the opportunities of using mRNA-LNPs in oncology applications and discuss the challenges for successfully translating the findings of preclinical studies of these nanoparticles into the clinic. We critically appraise the potential of various mRNA-LNP targeting and delivery strategies, considering physiological, technological and manufacturing challenges. We explore these approaches in the context of the potential clinical applications best suited to each approach and highlight the obstacles that currently need to be addressed to achieve these applications. Finally, we provide insights from preclinical and clinical studies that are leading to this powerful platform being considered the next frontier in oncology treatment.

A single-dose F1-based mRNA-LNP vaccine provides protection against the lethal plague bacterium.

Messenger RNA (mRNA) lipid nanoparticle (LNP) vaccines have emerged as an effective vaccination strategy. Although currently applied toward viral pathogens, data concerning the platform's effectiveness against bacterial pathogens are limited. Here, we developed an effective mRNA-LNP vaccine against a lethal bacterial pathogen by optimizing mRNA payload guanine and cytosine content and antigen design. We designed a nucleoside-modified mRNA-LNP vaccine based on the bacterial F1 capsule antigen, a major protective component of Yersinia pestis, the etiological agent of plague. Plague is a rapidly deteriorating contagious disease that has killed millions of people during the history of humankind. Now, the disease is treated effectively with antibiotics; however, in the case of a multiple-antibiotic-resistant strain outbreak, alternative countermeasures are required. Our mRNA-LNP vaccine elicited humoral and cellular immunological responses in C57BL/6 mice and conferred rapid, full protection against lethal Y. pestis infection after a single dose. These data open avenues for urgently needed effective antibacterial vaccines.

Nanotechnology-based strategies against SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already infected more than 500 million people globally (as of May 2022), creating the coronavirus disease 2019 (COVID-19) pandemic. Nanotechnology has played a pivotal role in the fight against SARS-CoV-2 in various aspects, with the successful development of the two highly effective nanotechnology-based messenger RNA vaccines being the most profound. Despite the remarkable efficacy of mRNA vaccines against the original SARS-CoV-2 strain, hopes for quickly ending this pandemic have been dampened by the emerging SARS-CoV-2 variants, which have brought several new pandemic waves. Thus, novel strategies should be proposed to tackle the crisis presented by existing and emerging SARS-CoV-2 variants. Here, we discuss the SARS-CoV-2 variants from biological and immunological perspectives, and the rational design and development of novel and potential nanotechnology-based strategies to combat existing and possible future SARS-CoV-2 variants. The lessons learnt and design strategies developed from this battle against SARS-CoV-2 variants could also inspire innovation in the development of nanotechnology-based strategies for tackling other global infectious diseases and their future variants.

Principles for designing an optimal mRNA lipid nanoparticle vaccine.

mRNA Lipid nanoparticles (LNPs) have recently been propelled onto the center stage of therapeutic platforms due to the success of the SARS-CoV-2 mRNA LNP vaccines (mRNA-1273 and BNT162b2), with billions of mRNA vaccine doses already shipped worldwide. While mRNA vaccines seem like an overnight success to some, they are in fact a result of decades of scientific research. The advantage of mRNA-LNP vaccines lies in the modularity of the platform and the rapid manufacturing capabilities. However, there is a multitude of choices to be made when designing an optimal mRNA-LNP vaccine regarding efficacy, stability and toxicity. Herein, we provide a brief on what we consider to be the most important aspects to cover when designing mRNA-LNPs from what is currently known and how to optimize them. Lastly, we give our perspective on which of these aspects is most crucial and what we believe are the next steps required to advance the field.

Resveratrol Enhances mRNA and siRNA Lipid Nanoparticles Primary CLL Cell Transfection.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western populations. Therapies such as mRNA and siRNA encapsulated in lipid nanoparticles (LNPs) represent a clinically advanced platform and are utilized for a wide variety of applications. Unfortunately, transfection of RNA into CLL cells remains a formidable challenge and a bottleneck for developing targeted therapies for this disease. Therefore, we aimed to elucidate the barriers to efficient transfection of RNA-encapsulated LNPs into primary CLL cells to advance therapies in the future. To this end, we transfected primary CLL patient samples with mRNA and siRNA payloads encapsulated in an FDA-approved LNP formulation and characterized the transfection. Additionally, we tested the potential of repurposing caffeic acid, curcumin and resveratrol to enhance the transfection of nucleic acids into CLL cells. The results demonstrate that the rapid uptake of LNPs is required for successful transfection. Furthermore, we demonstrate that resveratrol enhances the delivery of both mRNA and siRNA encapsulated in LNPs into primary CLL patient samples, overcoming inter-patient heterogeneity. This study points out the important challenges to consider for efficient RNA therapeutics for CLL patients and advocates the use of resveratrol in combination with RNA lipid nanoparticles to enhance delivery into CLL cells.

Immune checkpoint inhibitor combinations: Current efforts and important aspects for success.

Immune checkpoint inhibitors (ICI) have emerged as a remarkable treatment option for diverse cancer types. Currently, ICIs are approved for an expanding array of cancer indications. However, the majority of patients still do not demonstrate a durable long-term response following ICI therapy. In addition, many patients receiving ICI therapy develop immune-related adverse events (irAEs) affecting a wide variety of organs. To increase the percentage of patients who benefit from ICI therapy and to reduce the occurrence of irAEs, there is an ongoing effort to combine current ICIs with novel checkpoints inhibitors or other therapeutic approaches to achieve a synergistic effect which is larger than the sum of its parts. In this review we highlight the essential factors for more effective ICI combinations. We describe how the design of these strategies should be driven by the tumor's immunological context. We analyze current combination strategies and describe how they can be improved to unleash the immune system's full anti-cancer potential as well as convert immunologically "cold" tumors into "hot" ones. We examine the efforts to combine current ICIs (PD-1 and CTLA-4) with novel checkpoints (TIM-3, LAG-3, VISTA, TIGIT and others), immunotherapies (CAR-T cells and Cancer Vaccines) and delivery strategies (bispecific antibodies and other delivery platforms). Importantly, we outline how can one optimally combine ICIs with traditional pillars of cancer therapy such as radiation therapy (RT) and chemotherapy. We discuss the considerations regarding successful combination with RT and chemotherapy; these include fractionation schemes and selection of chemotherapeutics which can both directly eradicate cancer cells as well as increase the infiltration of immune cells into tumors. Finally, we critically assess these approaches and attempt to establish their strengths and weaknesses based on pre-clinical and clinical data.

Therapeutic mRNA delivery to leukocytes.

In the past few years mRNA molecules have become an attractive class of new drugs with numerous opportunities. mRNA therapeutics holds great potential for the treatment of a variety of diseases and is an emerging field both in academia and in the pharma industry. Among the various mRNA-based therapeutic approaches, immunotherapy is one of the pioneering and most advanced therapy to date. Immune cells manipulation using mRNA is commonly used for vaccination purposes, but its potential is much broader. In this review, we discuss the promise of mRNA delivery to leukocytes. We aim to provide an overview of the main progress in this field, as well as the challenges in expanding the scope of mRNA therapy for different therapeutic applications. We also discuss the main technological developments for overcoming mRNA therapy limitations, including mRNA rational design and delivery platforms.