Non-viral Nanoparticles Research Articles

Ligand-free biodegradable poly(beta-amino ester) nanoparticles for targeted systemic delivery of mRNA to the lungs

Non-viral nanoparticles (NPs) have seen heightened interest as a delivery method for a variety of clinically relevant nucleic acid cargoes in recent years. While much of the focus has been on lipid NPs, non-lipid NPs, including polymeric NPs, have the possibility of improved efficacy, safety, and targeting, especially to non-liver organs following systemic administration. A safe and effective systemic approach for intracellular delivery to the lungs could overcome limitations to intratracheal/intranasal delivery of NPs and improve clinical benefit for a range of diseases including cystic fibrosis. Here, engineered biodegradable poly (beta-amino ester) (PBAE) NPs are shown to facilitate efficient delivery of mRNA to primary human airway epithelial cells from both healthy donors and individuals with cystic fibrosis. Optimized NP formulations made with differentially endcapped PBAEs and systemically administered in vivo lead to high expression of mRNA within the lungs in BALB/c and C57 B/L mice without requiring a complex targeting ligand. High levels of mRNA-based gene editing were achieved in an Ai9 mouse model across bronchial, epithelial, and endothelial cell populations. No toxicity was observed either acutely or over time, including after multiple systemic administrations of the NPs. The non-lipid biodegradable PBAE NPs demonstrate high levels of transfection in both primary human airway epithelial cells and in vivo editing of lung cell types that are targets for numerous life-limiting diseases particularly single gene disorders such as cystic fibrosis and surfactant deficiencies.

Application of therapeutic nucleic acids and RNA interference to create products for personalised medicine

INTRODUCTION. Small interfering RNAs (siRNAs) are among the most promising types of therapeutic nucleic acids aiming at the inhibition of pathogenetically relevant gene expression through the RNA interference mechanism. However, the limited bioavailability and immunogenicity of siRNAs and imperfect delivery systems hinder the clinical potential and applicability of siRNA medicinal products.AIM. This study aimed to summarise recent advances in the development of siRNA medicinal products and the corresponding delivery systems, review clinical trial results, and outline future development prospects for these medicinal products.DISCUSSION. This article covers the molecular mechanisms underlying RNA interference, the considerations for siRNA development, and the techniques for effective siRNA delivery. The article dwells upon various systems for nucleic acid delivery to targeted cells. The most promising delivery systems are non-viral systems, including liposomes, exosomes, nanoparticles, polymers, cell-penetrating peptides, and GalNAc ligands. Their main advantages include their ease of complexation with nucleic acids, modification and functionalization potential, favourable safety profile, ability to cross biological barriers, and tropism to target tissues. The article summarises the information that has accumulated over the past few years in clinical trials of siRNA medicinal products for a range of conditions, including metabolic disorders, infections, and cancers, as well as hereditary, ophthalmic, renal, and hepatic diseases. Special attention is paid to siRNA medicinal products undergoing clinical trials (over 10 products) and approved for clinical use (6 products, including MIR 19, the first authorised Russian siRNA medicinal product).CONCLUSION. Ultimately, siRNA medicinal products are a promising tool for personalised medicine, exhibiting therapeutic potential for a wide range of pathological conditions. Further studies of siRNA medicinal products should aim at improving siRNA production technology to increase their bioavailability and half-life period. In addition, these studies should aim at enhancing delivery systems for these products to mitigate toxicity risks and maximise efficacy.

Suprachoroidal gene transfer with nonviral nanoparticles in large animal eyes.

Suprachoroidal nonviral gene therapy with biodegradable poly(β-amino ester) nanoparticles (NPs) provides widespread expression in photoreceptors and retinal pigmented epithelial (RPE) cells and therapeutic benefits in rodents. Here, we show in a human-sized minipig eye that suprachoroidal injection of 50 μl of NPs containing 19.2 μg of GFP expression plasmid caused GFP expression in photoreceptors and RPE throughout the entire eye with no toxicity. Two weeks after injection of 50, 100, or 200 μl, there was considerable within-eye and between-eye variability in expression that was reduced 3 months after injection of 200 μl and markedly reduced after three suprachoroidal injections at different locations around the eye. Reduction of bacterial CpG sequences in the expression plasmid resulted in a trend toward higher expression. These data indicate that nonviral suprachoroidal gene therapy with optimized polymer, expression plasmid, and injection approach has potential for treating photoreceptors throughout the entire retina of a human-sized eye.

CaCO3 Nanoparticles Delivering MicroRNA-200c Suppress Oral Squamous Cell Carcinoma

MicroRNA (miR)–200c suppresses the initiation and progression of oral squamous cell carcinoma (OSCC), the most prevalent head and neck cancer with high recurrence, metastasis, and mortality rates. However, miR-200c–based gene therapy to inhibit OSCC growth has yet to be reported. To develop an miR-based gene therapy to improve the outcomes of OSCC treatment, this study investigates the feasibility of plasmid DNA (pDNA) encoding miR-200c delivered via nonviral CaCO3-based nanoparticles to inhibit OSCC tumor growth. CaCO3-based nanoparticles with various ratios of CaCO3 and protamine sulfate (PS) were used to transfect pDNA encoding miR-200c into OSCC cells, and the efficiency of these nanoparticles was evaluated. The proliferation, migration, and associated oncogene production, as well as in vivo tumor growth for OSCC cells overexpressing miR-200c, were also quantified. It was observed that, while CaCO3-based nanoparticles improve transfection efficiencies of pDNA miR-200c, the ratio of CaCO3 to PS significantly influences the transfection efficiency. Overexpression of miR-200c significantly reduced proliferation, migration, and oncogene expression of OSCC cells, as well as the tumor size of cell line–derived xenografts (CDX) in mice. In addition, a local administration of pDNA miR-200c using CaCO3 delivery significantly enhanced miR-200c transfection and suppressed tumor growth of CDX in mice. These results strongly indicate that the nanocomplexes of CaCO3/pDNA miR-200c may potentially be used to reduce oral cancer recurrence and improve clinical outcomes in OSCC treatment, while more comprehensive examinations to confirm the safety and efficacy of the CaCO3/pDNA miR-200c system using various preclinical models are needed.

Non-Viral RNA-Lipid Nanoparticles for High Efficiency Genome Editing of CD34+ Hematopoietic Stem and Progenitor Cells for Advanced Cell and Gene Therapies

Background and Aims: The application of CRISPR-Cas9 gene editing in hematopoietic stem (HSC) and progenitor cells (HSPCs) has the potential to revolutionize the treatment of genetic blood disorders by correcting disease-causing mutations. Previously, we demonstrated the utility of a novel lipid nanoparticle (LNP) reagent for the multiplexed engineering of gene-edited CAR T cells, showing high cell viabilities, rapid onset editing, and potent CAR-mediated killing. In this current work, we demonstrate the capability of LNPs for the efficient and gentle delivery of genetic material to CD34+ HSPCs. The benefits of a non-viral LNP-mediated approach paves the way for the development of next generation gene therapy products in the HSC field. Methods: LNPs encapsulating S.p. Cas9 mRNA and CD33 or CD45 targeted guide RNA (sgRNA) were produced using the scalable NanoAssemblr® NxGen™ microfluidic platform technology ( Fig. 1A). The LNP composition was optimized for the gentle and efficient cargo delivery to HSC/HSPCs. Purified human CD34+ cells were cultured, stimulated, and treated by the direct addition of the RNA-LNPs. Various commercial cell sources were tested including mobilized peripheral blood and cord blood. Gene knockout and viability were assessed using flow cytometry, cell proliferation using an automated cell counter, and differentiation capacity by colony-forming unit (CFU) assays. Results: One-step addition of LNPs to CD34+ HSPCs resulted in 84 ± 6% CD33 and 81 ± 2% CD45 knockout efficiencies (n=6 CD34+ donors, Fig. 1B). After treatment, cells maintained on average 95 ± 3% absolute cell viability, compared to 99% viability of the untreated cells, Fig. 1C. Furthermore, LNP treated HSCs maintained excellent cell proliferation, with over >90% relative cell proliferation to the untreated controls. The CFU assays showed no significant change in relative lineage formation for both the RNA-LNPs and the empty LNP vehicle control ( Fig. 1D). Finally, LNP production was successfully scaled-up using microfluidics from discovery to pre-clinical scales. Conclusions: LNP-mediated CRISPR-Cas9 mRNA delivery is a promising approach for gene editing in HSPCs. The simple and gentle nature of LNP cell treatment allows for multiple genetic engineering steps for simultaneous expression and deletion of proteins for novel gene therapies. Furthermore, LNPs can be easily manufactured using microfluidics, enabling small-scale screening of RNA libraries and rapid scale-up for clinical translation.

Biomaterial‐Based Gene Delivery to Central Nervous System Cells for the Treatment of Spinal Cord Injury

Spinal cord injury (SCI) is a devastating traumatic injury often causing permanent loss of function. The challenge of treating SCI stems from the development of a complex pathophysiology at the site of injury, involving multiple biochemical cascades, widespread inflammation, blood supply interruption, inhibitory scar formation, and poor regrowth of injured axons. Clinical options are limited to surgical stabilization and attempt to ameliorate secondary damage following injury. Gene therapy has significant potential to tackle multiple aspects of SCI and improve functional outcomes. The emergence of a diverse array of biomaterial‐based nonviral nanoparticle vectors capable of delivering gene‐modifying nucleic acids offers the potential to improve the efficiency and specificity of genetic cargos for spinal cord regeneration. In this review, the progress that has been made in the field of SCI repair and the different types of nanoparticles and nucleic acid cargoes that have been used are outlined, placing a particular focus on the different cell types and pathways targeted. While many challenges remain, a perspective on the future of the field of nanoparticle‐mediated gene delivery for SCI is provided, including using biomaterial scaffolds engineered specifically for SCI to deliver gene therapeutics and the exciting opportunities that exist in the post‐COVID landscape.

Assessment of Different Niosome Formulations for Optogenetic Applications: Morphological and Electrophysiological Effects.

Gene therapy and optogenetics are becoming promising tools for treating several nervous system pathologies. Currently, most of these approaches use viral vectors to transport the genetic material inside the cells, but viruses present some potential risks, such as marked immunogenicity, insertional mutagenesis, and limited insert gene size. In this framework, non-viral nanoparticles, such as niosomes, are emerging as possible alternative tools to deliver genetic material, avoiding the aforementioned problems. To determine their suitability as vectors for optogenetic therapies in this work, we tested three different niosome formulations combined with three optogenetic plasmids in rat cortical neurons in vitro. All niosomes tested successfully expressed optogenetic channels, which were dependent on the ratio of niosome to plasmid, with higher concentrations yielding higher expression rates. However, we found changes in the dendritic morphology and electrophysiological properties of transfected cells, especially when we used higher concentrations of niosomes. Our results highlight the potential use of niosomes for optogenetic applications and suggest that special care must be taken to achieve an optimal balance of niosomes and nucleic acids to achieve the therapeutic effects envisioned by these technologies.

Lipid nanoparticle topology regulates endosomal escape and delivery of RNA to the cytoplasm.

RNA therapeutics have the potential to resolve a myriad of genetic diseases. Lipid nanoparticles (LNPs) are among the most successful RNA delivery systems. Expanding their use for the treatment of more genetic diseases hinges on our ability to continuously evolve the design of LNPs with high potency, cellular-specific targeting, and low side effects. Overcoming the difficulty of releasing cargo from endocytosed LNPs remains a significant hurdle. Here, we investigate the fundamental properties of nonviral RNA nanoparticles pertaining to the activation of topological transformations of endosomal membranes and RNA translocation into the cytosol. We show that, beyond composition, LNP fusogenicity can be prescribed by designing LNP nanostructures that lower the energetic cost of fusion and fusion-pore formation with a target membrane. The inclusion of structurally active lipids leads to enhanced LNP endosomal fusion, fast evasion of endosomal entrapment, and efficacious RNA delivery. For example, conserving the lipid make-up, RNA-LNPs having cuboplex nanostructures are significantly more efficacious at endosomal escape than traditional lipoplex constructs.

Gene modification therapies for hereditary diseases in the fetus.

Proof-of-principle disease models have demonstrated the feasibility of an intrauterine gene modification therapy (in utero gene therapy (IUGT)) approach to hereditary diseases as diverse as coagulation disorders, haemoglobinopathies, neurogenetic disorders, congenital metabolic, and pulmonary diseases. Gene addition, which requires the delivery of an integrating or episomal transgene to the target cell nucleus to be transcribed, and gene editing, where the mutation is corrected within the gene of origin, have both been used successfully to increase normal protein production in a bid to reverse or arrest pathology in utero. While most experimental models have employed lentiviral, adenoviral, and adeno-associated viral vectors engineered to efficiently enter target cells, newer models have also demonstrated the applicability of non-viral lipid nanoparticles. Amelioration of pathology is dependent primarily on achieving sustained therapeutic transgene expression, silencing of transgene expression, production of neutralising antibodies, the dilutional effect of the recipient's growth on the mass of transduced cells, and the degree of pre-existing cellular damage. Safety assessment of any IUGT strategy will require long-term postnatal surveillance of both the fetal recipient and the maternal bystander for cell and genome toxicity, oncogenic potential, immune-responsiveness, and germline mutation. In this review, we discuss advances in the field and the push toward clinical translation of IUGT.

Hydrogels for RNA delivery.

RNA-based therapeutics have shown tremendous promise in disease intervention at the genetic level, and some have been approved for clinical use, including the recent COVID-19 messenger RNA vaccines. The clinical success of RNA therapy is largely dependent on the use of chemical modification, ligand conjugation or non-viral nanoparticles to improve RNA stability and facilitate intracellular delivery. Unlike molecular-level or nanoscale approaches, macroscopic hydrogels are soft, water-swollen three-dimensional structures that possess remarkable features such as biodegradability, tunable physiochemical properties and injectability, and recently they have attracted enormous attention for use in RNA therapy. Specifically, hydrogels can be engineered to exert precise spatiotemporal control over the release of RNA therapeutics, potentially minimizing systemic toxicity and enhancing in vivo efficacy. This Review provides a comprehensive overview of hydrogel loading of RNAs and hydrogel design for controlled release, highlights their biomedical applications and offers our perspectives on the opportunities and challenges in this exciting field of RNA delivery.

Packaging and Uncoating of CRISPR/Cas Ribonucleoproteins for Efficient Gene Editing with Viral and Non-Viral Extracellular Nanoparticles

Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized functional genomic studies and genetic disease correction. While numerous gene editing applications have been easily adapted by experimental science, the clinical utility of CRISPR/Cas remains very limited due to difficulty in delivery to primary cells and possible off-target effects. The use of CRISPR in the form of a ribonucleoprotein (RNP) complex substantially reduces the time of DNA exposure to the effector nuclease and minimizes its off-target activity. The traditional electroporation and lipofection methods lack the cell-type specificity of RNP delivery, can be toxic for cells, and are less efficient when compared to nanoparticle transporters. This review focuses on CRISPR/Cas RNP packaging and delivery using retro/lentiviral particles and exosomes. First, we briefly describe the natural stages of viral and exosomal particle formation, release and entry into the target cells. This helps us understand the mechanisms of CRISPR/Cas RNP packaging and uncoating utilized by the current delivery systems, which we discuss afterward. Much attention is given to the exosomes released during viral particle production that can be passively loaded with RNPs as well as the mechanisms necessary for particle fusion, RNP release, and transportation inside the target cells. Collectively, together with specific packaging mechanisms, all these factors can substantially influence the editing efficiency of the system. Finally, we discuss ways to improve CRISPR/Cas RNP delivery using extracellular nanoparticles.

Stimuli-Responsive Non-viral Nanoparticles for Gene Delivery.

Considering nucleic acids as the language of life and the genome as the instruction manual of cells, their targeted modulation promises great opportunities in treating and healing diseases. In addition to viral gene transfer, the overwhelming power of non-viral mRNA-based vaccines is driving the development of novel gene transporters. Thereby, various nucleic acids such as DNA (pDNA) or RNA (mRNA, siRNA, miRNA, gRNA, or ASOs) need to be delivered, requiring a transporter due to their high molar mass and negative charge in contrast to classical agents. This chapter presents the specific biological hurdles for using nucleic acids and shows how new materials can overcome these.

Nonviral nanoparticle gene delivery into the CNS for neurological disorders and brain cancer applications.

Nonviral nanoparticles have emerged as an attractive alternative to viral vectors for gene therapy applications, utilizing a range of lipid-based, polymeric, and inorganic materials. These materials can either encapsulate or be functionalized to bind nucleic acids and protect them from degradation. To effectively elicit changes to gene expression, the nanoparticle carrier needs to undergo a series of steps intracellularly, from interacting with the cellular membrane to facilitate cellular uptake to endosomal escape and nucleic acid release. Adjusting physiochemical properties of the nanoparticles, such as size, charge, and targeting ligands, can improve cellular uptake and ultimately gene delivery. Applications in the central nervous system (CNS; i.e., neurological diseases, brain cancers) face further extracellular barriers for a gene-carrying nanoparticle to surpass, with the most significant being the blood-brain barrier (BBB). Approaches to overcome these extracellular challenges to deliver nanoparticles into the CNS include systemic, intracerebroventricular, intrathecal, and intranasal administration. This review describes and compares different biomaterials for nonviral nanoparticle-mediated gene therapy to the CNS and explores challenges and recent preclinical and clinical developments in overcoming barriers to nanoparticle-mediated delivery to the brain. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Incorporation of phosphatidylserine improves efficiency of lipid based gene delivery systems

The essential homeostatic process of dead cell clearance (efferocytosis) is used by viruses in an act of apoptotic mimicry. Among others, virions leverage phosphatidylserine (PS) as an essential “eat me” signal in viral envelopes to increase their infectivity. In a virus-inspired biomimetic approach, we demonstrate that PS can be incorporated into non-viral lipid nanoparticle (LNP) pDNA/mRNA constructs to enhance cellular transfection. The inclusion of the bioactive PS leads to an increased ability of LNPs to deliver nucleic acids in vitro to cultured HuH-7 hepatocellular carcinoma cells resulting in a 6-fold enhanced expression of a transgene. Optimal PS concentrations are in the range of 2.5 to 5% of total lipids. PS-decorated mRNA-LNPs show a 5.2-fold enhancement of in vivo transfection efficiency as compared to mRNA-LNPs devoid of PS. Effects were less pronounced for PS-decorated pDNA-LNPs (3.2-fold increase). Incorporation of small, defined amounts of PS into gene delivery vectors opens new avenues for efficient gene therapy and can be easily extended to other therapeutic systems.

Development of Delivery Systems for Local Administration of Cytokines/Cytokine Gene-Directed Therapeutics: Modern Oncologic Implications.

In this review, we discuss modern cytokine delivery systems in oncologic care, focusing on modalities being developed in the clinical trials or currently in use. These include pegylation, immune-cytokine drug conjugates, cytokine-expressing plasmid nanoparticles, nonviral cytokine nanoparticles, viral systems, and AcTakines. Cytokine therapy has the potential to contribute to cancer treatment options by modulating the immune system towards an improved antitumor response and has shown promise both independently and in combination with other immunotherapy agents. Despite promising preliminary studies, systemic toxicities and challenges with administration have limited the impact of unmodified cytokine therapy. In the last decade, novel delivery systems have been developed to address these challenges and facilitate cytokine-based oncologic treatments. Novel delivery systems provide potential solutions to decrease dose-limiting side effects, facilitate administration, and increase the therapeutic activity of cytokine treatments in oncology care. The expanding clinical and translational research in these systems provides an opportunity to augment the armamentarium of immune oncology and may represent the next frontier of cytokine-based immuno-oncology.

Controlled pVEGF delivery via a gene-activated matrix comprised of a peptide-modified non-viral vector and a nanofibrous scaffold for skin wound healing

Regulating cell function and tissue formation by combining gene delivery with functional scaffolds to create gene-activated matrices (GAMs) is a promising strategy for tissue engineering. However, fabrication of GAMs with low cytotoxicity, high transfection efficiency, and long-term gene delivery properties remains a challenge. In this study, a non-viral DNA delivery nanocomplex was developed by modifying poly (D, L-lactic-co-glycolic acid)/polyethylenimine (PLGA/PEI) nanoparticles with the cell-penetrating peptide KALA. Subsequently, the nanocomplex carrying plasmid DNA encoding vascular endothelial growth factor (pVEGF) was immobilized onto a polydopamine-coated electrospun alginate nanofibrous scaffold, resulting in a GAM for enhanced skin wound healing. The nanocomplex exhibited much lower cytotoxicity and comparable or even higher transfection efficiency compared with PEI. The GAM enabled sustained gene release and long-tern transgene expression of VEGF in vitro. In an excisional full-thickness skin wound rat model, the GAM could accelerate wound closure, promote complete re-epithelization, reduce inflammatory response, and enhance neovascularization, ultimately enhancing skin wound healing. The current GAM comprising a low-toxic gene delivery nanocomplex and a biocompatible 3D nanofibrous scaffold demonstrates great potential for mediating long-term cell functions and may become a powerful tool for gene delivery in tissue engineering. Statement of significanceGene delivery is a promising strategy in promoting tissue regeneration as an effective alternative to growth factor delivery, but the study on three-dimensional gene-activated scaffolds remains in its infancy. Herein, a biodegradable nanofibrous gene-activated matrix integrating non-viral nanoparticle vector was designed and evaluated both in vitro and in vivo. The results show that the nanoparticle vector provided high transfection efficiency with minimal cytotoxicity. After surface immobilization of the nanocomplexes carrying plasmid DNA encoding vascular endothelial growth factor (pVEGF), the nanofibrous scaffold enabled sustained DNA release and long-term transgene expression in vitro. In a rat full-thickness skin wound model, the scaffold could accelerate wound healing. This innovative gene-activated matrix can be a promising candidate for tissue regeneration.

Developing Biodegradable Lipid Nanoparticles for Intracellular mRNA Delivery and Genome Editing.

Since the U.S. Food and Drug Administration (FDA) granted emergency use authorization for two mRNA vaccines against SARS-CoV-2, mRNA-based technology has attracted broad attention from the scientific community to investors. When delivered intracellularly, mRNA has the ability to produce various therapeutic proteins, enabling the treatment of a variety of illnesses, including but not limited to infectious diseases, cancers, and genetic diseases. Accordingly, mRNA holds significant therapeutic potential and provides a promising means to target historically hard-to-treat diseases. Current clinical efforts harnessing mRNA-based technology are focused on vaccination, cancer immunotherapy, protein replacement therapy, and genome editing. The clinical translation of mRNA-based technology has been made possible by leveraging nanoparticle delivery methods. However, the application of mRNA for therapeutic purposes is still challenged by the need for specific, efficient, and safe delivery systems.This Account highlights key advances in designing and developing combinatorial synthetic lipid nanoparticles (LNPs) with distinct chemical structures and properties for in vitro and in vivo intracellular mRNA delivery. LNPs represent the most advanced nonviral nanoparticle delivery systems that have been extensively investigated for nucleic acid delivery. The aforementioned COVID-19 mRNA vaccines and one LNP-based small interfering RNA (siRNA) drug (ONPATTRO) have received clinical approval from the FDA, highlighting the success of synthetic ionizable lipids for in vivo nucleic acid delivery. In this Account, we first summarize the research efforts from our group on the development of bioreducible and biodegradable LNPs by leveraging the combinatorial chemistry strategy, such as the Michael addition reaction, which allows us to easily generate a large set of lipidoids with diverse chemical structures. Next, we discuss the utilization of a library screening strategy to identify optimal LNPs for targeted mRNA delivery and showcase the applications of the optimized LNPs in cell engineering and genome editing. Finally, we outline key challenges to the clinical translation of mRNA-based therapies and propose an outlook for future directions of the chemical design and optimization of LNPs to improve the safety and specificity of mRNA drugs. We hope this Account provides insight into the rational design of LNPs for facilitating the development of mRNA therapeutics, a transformative technology that promises to revolutionize future medicine.

Nucleic acid delivery and nanoparticle design for COVID vaccines.

Nucleic acid therapeutics offer a new paradigm to rapidly respond to global health problems. The versatility of nucleic acids, especially in RNA therapies, provides the ability to tune levels of specific protein expression, achieving downregulation through short interfering RNA (siRNA) or upregulation by messenger RNA (mRNA) administration. Recent advances in the development of delivery vehicles, including nonviral nanoparticles are crucial to overcome the innate barriers to nucleic acid delivery. Toward this end, current clinical approaches have utilized mRNA and lipid nanoparticles (LNPs) to address the COVID-19 pandemic through novel vaccine strategies, producing efficacious vaccines within one year of sequencing the SARS-CoV-2 genome. Here, we review fundamental concepts required to achieve successful nucleic acid delivery, including the design of LNP systems optimized for mRNA vaccine applications.Graphic abstract

Reasons for success and lessons learnt from nanoscale vaccines against COVID-19.

Almost all currently used vaccines against COVID-19 consist of either non-viral or viral nanoparticles. Here we attempt to understand the reasons behind the success of such advanced nanoscale vaccine technologies compared with clinically established conventional vaccines, and the lessons to be learnt from this potentially transformative development in the adoption and acceptance of nanotechnology for medicine.

Peptide-Based Nanoparticles for Therapeutic Nucleic Acid Delivery.

Gene therapy offers the possibility to skip, repair, or silence faulty genes or to stimulate the immune system to fight against disease by delivering therapeutic nucleic acids (NAs) to a patient. Compared to other drugs or protein treatments, NA-based therapies have the advantage of being a more universal approach to designing therapies because of the versatility of NA design. NAs (siRNA, pDNA, or mRNA) have great potential for therapeutic applications for an immense number of indications. However, the delivery of these exogenous NAs is still challenging and requires a specific delivery system. In this context, beside other non-viral vectors, cell-penetrating peptides (CPPs) gain more and more interest as delivery systems by forming a variety of nanocomplexes depending on the formulation conditions and the properties of the used CPPs/NAs. In this review, we attempt to cover the most important biophysical and biological aspects of non-viral peptide-based nanoparticles (PBNs) for therapeutic nucleic acid formulations as a delivery system. The most relevant peptides or peptide families forming PBNs in the presence of NAs described since 2015 will be presented. All these PBNs able to deliver NAs in vitro and in vivo have common features, which are characterized by defined formulation conditions in order to obtain PBNs from 60 nm to 150 nm with a homogeneous dispersity (PdI lower than 0.3) and a positive charge between +10 mV and +40 mV.