Figure 1 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Schematic illustration of CRISPR/Cas9-mediated gene editing in eukaryotic cells. The CRISPR/Cas9 protein utilizes both sgRNA and PAM to recognize target DNA and induces DSB. This process triggers NHEJ-or HDR-mediated targeted genome editing. Created with BioRender.com.
Source publication
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated (Cas) genome editing system provides a powerful toolbox for genetic engineering. However, safe and efficient in vivo delivery of all CRISPR components into the nuclei of target organs remains challenging toward CRISPR/Cas clinical translation. With the rapid de...
Citations
... Viral [28] and non-viral [29-31] vectors are common strategies for delivering nucleic acids to desired sites. However, viral delivery has limitations due to (i) inherent toxicity, (ii) triggering immune responses [1], and (iii) high costs due to difficulty in scaling up production [32]. There has been extensive effort devoted to developing non-viral vectors, especially those based on liposomes, lipid nanoparticles, polypeptides, protamines, polymers, micelles, inorganic-based materials, and hybrid systems. ...
Single-stranded messenger ribonucleic acid (mRNA) plays a pivotal role in transferring genetic information, and tremendous effort has been devoted over the years to utilize its transcription efficacy in therapeutic interventions for a variety of diseases with high morbidity and mortality. Lipid nanocarriers have been extensively investigated for mRNA delivery and enabled the rapid and successful development of mRNA vaccines against SARS-CoV-2. Some constraints of lipid nanocarriers have encouraged the development of alternative delivery systems, such as polymer-based soft nanoparticles, which offer a modular gene delivery platform. Such macromolecule-based nanocarriers can be synthetically articulated for tailored parameters including mRNA protection, loading efficacy, and targeted release. In this review, we highlight recent advances in the development of polymeric architectures for mRNA delivery, their limitations, and the challenges that still exist, with the aim of expediting further research and the clinical translation of such formulations.
... Lipid nanoparticles (LNPs) represent advanced nucleic acid delivery systems that have been used for clinically approved siRNA-and mRNA-based therapeutics [1][2][3][4][5][6]. The vast majority of LNPs that are already being used in preclinical and clinical trials, however, are accumulated in the liver following intravenous administration, limiting their further therapeutic applications [4][5][6][7][8][9][10][11][12][13]. The most prevalent explanation on the mechanisms of hepatic tropism is that LNPs adsorb apolipoprotein E during circulation in blood and boost internalization by hepatocytes via low-density lipoprotein receptor (LDLR)-mediated endocytosis [14][15][16][17][18][19]. ...
Background: As the overwhelming majority of advanced mRNA delivery systems are preferentially accumulated in the liver, there is an accelerating growth in the demand for the development of non-liver mRNA delivery platforms.
Methods: In this study, we prepared cationic lipid-like nanoassemblies through a N-quaternizing strategy. Their physicochemical properties, in vitro mRNA delivery efficiency, and organ tropism in mice were investigated.
Results: Introduction of quaternary ammonium groups onto lipid-like nanoassemblies not only enhances their mRNA delivery performance in vitro, but also completely alters their tropism from the spleen to the lung after intravenous administration in mice. Quaternized lipid-like nanoassemblies exhibit ultra-high specificity to the lung and are predominantly taken up by pulmonary immune cells, leading to over 95% of exogenous mRNA translation in the lungs. Such mRNA delivery carriers are stable even after more than one-year storage at ambient temperature.
Conclusions: Quaternization provides an alternative method for design of new lung-targeted mRNA delivery systems without incorporation of targeting ligands, which should extend the therapeutic applicability of mRNA to lung diseases.
... Off-target effects are genetic alterations to nontargeted regions of the genome that may have detrimental impacts. Several approaches have been created to limit off-target effects in attempt to overcome this issue, including better CRISPR-Cas9 designs (Huang et al. 2022 ), enhanced guide RNAs Yin 2019 , Hu et al. 2023 ), and improved delivery methods (Liu et al. 2022c ). Advanced computational tools have facilitated off-target prediction and continuous progress has been reported in its precision (Li et al. 2022a ). ...
Nutraceuticals are defined as food or food components with therapeutic capabilities that have few side effects and are regarded as a natural therapy for preventing the onset of several life-threatening illnesses. The use of microbial cell factories to produce nutraceuticals is considered to be sustainable and promising for meeting market demand. Among the diverse strategies for optimizing microbial cell factories, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system has emerged as a valuable tool for gene integration, deletion, activation, and downregulation. With the advent of multiplexed and precise CRISPR strategies, optimized microbial cell factories are revolutionizing the yield of nutraceuticals. This review focuses on the development of highly adaptable CRISPR strategies to optimize the production in microbial cell factories of some important nutraceuticals (belonging to the class of carotenoids, flavonoids, stilbenoids, polysaccharides, and non-protein amino acids). Further, we highlighted current challenges related to the efficiency of CRISPR strategies and addressed potential future directions to fully harness CRISPR strategies to make nutraceutical synthesis in microbial cell factories an industrially favorable method.
... [81][82][83] In contrast, delivering CRISPR RNA by using LNPs might provide cellselective targeting and enable genome editing in specific cell types, thus reducing the risk of off-target effects. [84] To increase the specificity of CRISPR RNA delivery and reduce off-target effects, recent studies have focused on the design of biodegradable LNPs with cell-selective targeting. For example, the introduction of disulfide bonds into the hydrophobic tail of ionizable lipids has been shown to promote endosomal escape and intracellular release of Cas9 mRNA, while also allowing for degradation under the action of intracellular glutathione. ...
Messenger RNA (mRNA) is being used as part of an emerging class of biotherapeutics with great promise for preventing and treating a wide range of diseases, as well as encoding programmable nucleases for genome editing. However, mRNA's low stability and immunogenicity, as well as the impermeability of the cell membrane to mRNA greatly limit mRNA's potential for therapeutic use. Lipid nanoparticles (LNPs) are currently one of the most extensively studied nanocarriers for mRNA delivery and have recently been clinically approved for developing mRNA‐based vaccines to prevent COVID‐19. In this review, we summarize the latest advances in designing ionizable lipids and formulating LNPs for intracellular and tissue‐targeted mRNA delivery. Furthermore, we discuss the progress of intracellular mRNA delivery for spatiotemporally controlled CRISPR/Cas9 genome editing by using LNPs. Finally, we provide a perspective on the future of LNP‐based mRNA delivery for CRISPR/Cas9 genome editing and the treatment of genetic disorders.
... These progresses have paved the road for mRNA as a new class of drug [2,[5][6][7]9,10]. Nowadays, mRNA-based therapeutics have displayed broad applications in prophylactic vaccines, protein replacement therapy, cancer immunotherapy, and gene editing [9][10][11][12][13][14]. ...
The clinical translation of messenger mRNA (mRNA)-based therapeutics requires safe and effective delivery systems. Although considerable progress has been made on the development of mRNA delivery systems, many challenges, such as the dose-limiting toxicity and specific delivery to extrahepatic tissues, still remain. Cell-derived vesicles, a type of endogenous membranous particle secreted from living cells, can be leveraged to load mRNA during or after their biogenesis. Currently, they have received increasing interest for mRNA delivery due to their natural origin, good biocompatibility, cell-specific tropism, and unique ability to cross physiological barriers. In this review, we provide an overview of recent advances in the naturally occurring mRNA delivery platforms and their biomedical applications. Furthermore, the future perspectives on clinical translation of cell-derived vesicles have been discussed.
Abstract: The concept of gene therapy has been around for over a hundred years. However, the research and development necessary to prove the efficacy and safety of gene therapy are tremendously time-consuming and require huge investments. Moreover, the application of gene therapy depends on the management techniques of genetic materials, which have advanced rapidly just recently. The history of clinical trials on gene therapy from the past to the present reveals both successes and failures. Correspondingly, a clinical trial on gene therapy has to be approved by a scientific review committee and an ethics committee to ensure that the benefits outweigh the risks. At present, there are several approved gene therapy products for the treatment of some diseases, while many products are currently undergoing late-phase clinical trials or waiting for approval. The development of gene therapy is adapted accordingly to innovations such as CRISPR/Cas9 gene editing that enable the utilization and modernization of gene therapy. In this review, we describe the concepts and principles of gene therapy, namely, the definition, key events in the history of gene therapy, therapeutic indications, type of gene therapy, strategy of gene therapy, ex vivo and in vivo gene therapy, delivery systems, immune responses, and adverse drug reactions, and lastly, we address some concerns about ethical issues.
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, will also be introduced. Finally, our insights into the clinical and translational development of mRNA nano-drugs are presented.
COVID-19 mRNA vaccines represent a completely new category of vaccines and play a crucial role in controlling the COVID-19 pandemic. In this study, we have developed a PEG-lipid-free two-component mRNA vaccine (PFTCmvac) by formulating mRNA encoding the receptor binding domain (RBD) of SARS-CoV-2 into lipid-like nanoassemblies. Without using polyethylene glycol (PEG)-lipids, the self-assembled PFTCmvac forms thermostable nanoassemblies and exhibits a dose-dependent cellular uptake and membrane disruption, eventually leading to high-level protein expression in both mammalian cells and mice. Vaccination with PFTCmvac elicits strong humoral and cellular responses in mice, without evidence of significant adverse reactions. In addition, the vaccine platform does not trigger complement activation in human serum, even at a high serum concentration. Collectively, the PEG-lipid-free two-component nanoassemblies provide an alternative delivery technology for COVID-19 mRNA vaccines and opportunities for the rapid production of new mRNA vaccines against emerging infectious diseases.
The broad biomedical applications of messenger RNA (mRNA)-based therapeutics rely heavily on the rapid development of mRNA delivery systems. In this study, dual-component lipid nanoparticles (LNPs) were developed for intracellular delivery of mRNA to macrophages. By screening a panel of quaternary ammonium compounds with varying alkyl or aryl side chains, a cationic surfactant containing two hexadecyl tails was found to be effective for mRNA delivery with the assistance of fusogenic lipids. Further investigating structure alterations and optimization of formulation parameters, we found that a surfactant-derived ionizable lipid mixed with a fusogenic lipid was capable of condensing mRNA and self-assembling into LNPs. Without using the PEGylated lipid, the resulting LNPs were able to render the exogenous mRNA resistant to hydrolysis by nucleases and displayed excellent biocompatibility, along with the capacity to deliver mRNA to hard-to-transfect. Overall, the dual-component nanocarrier provides a novel mRNA delivery platform for genetic engineering of macrophages.
