bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2023–09–24
nineteen papers selected by
the Merkel lab, Ludwig-Maximilians University
  1. Differences and Similarities of the Intravenously Administered Lipid Nanoparticles in Three Clinical Trials: Potential Linkage between Lipid Nanoparticles and Extracellular Vesicles.
  2. Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery.
  3. Lipid Nanoparticles Deliver mRNA to the Brain after an Intracerebral Injection.
  4. Delivery of siRNA using cationic rosette nanotubes for gene silencing.
  5. mRNA lipid nanoparticles induce immune tolerance to treat human diseases.
  6. TAT decorated siRNA polyplexes for inhalation delivery in anti-asthma therapy.
  7. mRNA nanodelivery systems: targeting strategies and administration routes.
  8. Nonclinical safety assessment of an mRNA Covid-19 vaccine candidate following repeated administrations and biodistribution.
  9. Optimization of HPLCCAD method for simultaneous analysis of different lipids in lipid nanoparticles with analytical QbD.
  10. In vivo self-assembly and delivery of VEGFR2 siRNA-encapsulated small extracellular vesicles for lung metastatic osteosarcoma therapy.
  11. Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells.
  12. Lipid-Centric Design of Plasma Membrane-Mimicking Nanocarriers for Targeted Chemotherapeutic Delivery.
  13. Inhalation delivery of nucleic acid gene therapies in preclinical drug development.
  14. mRNA vaccines in disease prevention and treatment.
  15. Engineering extracellular vesicles to deliver CRISPR ribonucleoprotein for gene editing.
  16. mRNA vaccine quality analysis using RNA sequencing.
  17. Microfluidic synthesis of nanomaterials for biomedical applications.
  18. Intranasal mRNA-LNP vaccination protects hamsters from SARS-CoV-2 infection.
  19. RNA-based medicine: from molecular mechanisms to therapy.
  1. Mol Pharm. 2023 Sep 17.
    Differences and Similarities of the Intravenously Administered Lipid Nanoparticles in Three Clinical Trials: Potential Linkage between Lipid Nanoparticles and Extracellular Vesicles.
    Yuta Suzuki, Yuri Katsurada, Kenji Hyodo.
      Lipid nanoparticles (LNPs) are clinically validated drug-delivery carriers. However, clinical data on intravenously administered LNPs are limited compared with those on intramuscularly administered LNPs (mRNA vaccines against COVID-19). Here, we reviewed three clinically tested intravenously administered LNPs (patisiran, mRNA-1944, and NTLA-2001). We summarize the differences and similarities in their formulations, mechanisms of action, and pharmacokinetics profiles. In humans, patisiran and mRNA-1944 exhibited similar multiphasic pharmacokinetic profiles with a secondary peak in the RNA concentration. siRNA (patisiran) and mRNA (mRNA-1944) exhibited prolonged blood circulation and were detectable for more than 28 days after a single administration. We further summarize the basics of extracellular vesicles (EVs) and discuss the potential linkages between LNPs and EVs. This Review provides an understanding of the human clinical data of intravenous LNP formulations, which can be potentially explored to develop next-generation LNP-and EV-based drug delivery carriers.
    Keywords:  RNA therapeutics; drug delivery; extracellular vesicles; lipid nanoparticles; mRNA
    DOI:  https://doi.org/10.1021/acs.molpharmaceut.3c00547
  2. J Control Release. 2023 Sep 16. pii: S0168-3659(23)00610-7. [Epub ahead of print]
    Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery.
    Alessio Malfanti, Haider Sami, Anna Balasso, Giulia Marostica, Busra Arpac, Francesca Mastrotto, Giuseppe Mantovani, Elisa Cola, Martina Anton, Paolo Caliceti, Manfred Ogris, Stefano Salmaso.
      Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]-distearoyl glycerol (Arg4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCEfree-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCEfree-LPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCEfree-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCEfree-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell up-take of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomotropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.
    Keywords:  Cell penetration enhancers; Endosomal escape; Lipoplex engineering; Oligo-cationic condensing lipids
    DOI:  https://doi.org/10.1016/j.jconrel.2023.09.022
  3. Biochemistry. 2023 Sep 20.
    Lipid Nanoparticles Deliver mRNA to the Brain after an Intracerebral Injection.
    Jan Tuma, Yu-Ju Chen, Michael G Collins, Abhik Paul, Jie Li, Hesong Han, Rohit Sharma, Niren Murthy, Hye Young Lee.
      Neurological disorders are often debilitating conditions with no cure. The majority of current therapies are palliative rather than disease-modifying; therefore, new strategies for treating neurological disorders are greatly needed. mRNA-based therapeutics have great potential for treating such neurological disorders; however, challenges with delivery have limited their clinical potential. Lipid nanoparticles (LNPs) are a promising delivery vector for the brain, given their safer toxicity profile and higher efficacy. Despite this, very little is known about LNP-mediated delivery of mRNA into the brain. Here, we employ MC3-based LNPs and successfully deliver Cre mRNA and Cas9 mRNA/Ai9 sgRNA to the adult Ai9 mouse brain; greater than half of the entire striatum and hippocampus was found to be penetrated along the rostro-caudal axis by direct intracerebral injections of MC3 LNP mRNAs. MC3 LNP Cre mRNA successfully transfected cells in the striatum (∼52% efficiency) and hippocampus (∼49% efficiency). In addition, we demonstrate that MC3 LNP Cas9 mRNA/Ai9 sgRNA edited cells in the striatum (∼7% efficiency) and hippocampus (∼3% efficiency). Further analysis demonstrates that MC3 LNPs mediate mRNA delivery to multiple cell types including neurons, astrocytes, and microglia in the brain. Overall, LNP-based mRNA delivery is effective in brain tissue and shows great promise for treating complex neurological disorders.
    DOI:  https://doi.org/10.1021/acs.biochem.3c00371
  4. Biomater Sci. 2023 Sep 21.
    Delivery of siRNA using cationic rosette nanotubes for gene silencing.
    Uyen Ho, Mounir El-Bakkari, Aws Alshamsan, Jae-Young Cho, Takeshi Yamazaki, Usha D Hemraz, Hicham Fenniri.
      The quest for new therapeutic treatments for hereditary diseases has led to many advances in RNA interference (RNAi) and gene silencing. While this technique has the potential to address many problems, the key to its continued use is the development of effective delivery strategies that would reduce cellular toxicity and increase silencing efficiency. Rosette nanotubes (RNTs) are biomimetic supramolecular nanostructures formed through the self-assembly of hybrid guanine-cytosine (G∧C) DNA bases. Here, we used bioactive RNTs for siRNA delivery and gene silencing. Fifteen lysine-functionalized twin-G∧C motifs (KnT, n = 1 to 15) were synthesized using solid phase peptide synthesis to produce building blocks that self-assembled to produce cationic RNTs under physiological conditions. The intracellular uptake of siRNA delivered by the oligo-L-lysine RNTs was examined and it was found that the complexation of siRNA was affected by the cationic charges from the lysine residues and the length of RNTs formed, with the higher charged KnT RNTs delivering siRNA to the cells at a faster rate. In addition, by protecting siRNA from serum degradation, KnT RNTs were shown to deliver their cargo to the cells effectively via the endocytic pathway. A reduction in the expression (∼70%) of the target stat3 protein was observed during gene expression analysis in HCT116 and A549 cell lines.
    DOI:  https://doi.org/10.1039/d3bm01115a
  5. Med Rev (Berl). 2023 Apr;3(2): 180-183
    mRNA lipid nanoparticles induce immune tolerance to treat human diseases.
    Wei Cao, Tian Xia.
      Rapid developments in the coronavirus disease 2019 (COVID-19) mRNA vaccine showcased the power of lipid nanoparticle (LNP) delivery systems in fighting infectious diseases. In addition, mRNA therapeutics are also in development for cancer immunotherapy. Recently, mRNA therapy has been expanded to induce immune tolerance, the opposite of immune-boosting effects, to treat diseases involving enhanced immune responses including allergies and autoimmune diseases. mRNA LNPs have been used to treat peanut allergy by us and autoimmune experimental autoimmune encephalomyelitis by Ugur Sahin. It is expected that more and more research is going to delve into the immune tolerance field for allergies and autoimmune diseases, where effective therapies are in short supply.
    Keywords:  allergy; autoimmune diseases; mRNA lipid nanoparticle; nanomedicine
    DOI:  https://doi.org/10.1515/mr-2023-0008
  6. Eur J Pharm Sci. 2023 Sep 16. pii: S0928-0987(23)00210-5. [Epub ahead of print]190 106580
    TAT decorated siRNA polyplexes for inhalation delivery in anti-asthma therapy.
    Salvatore Emanuele Drago, Marta Cabibbo, Emanuela Fabiola Craparo, Gennara Cavallaro.
      In this work, a novel protonable copolymer was designed to deliver siRNA through the inhalation route, as an innovative formulation for the management of asthma. This polycation was synthesized by derivatization of α,β-poly(N-2-hydroxyethyl)D,L-aspartamide (PHEA) first with 1,2-Bis(3-aminopropylamino)ethane (bAPAE) and then with a proper amount of maleimide terminated poly(ethylene glycol) (PEG-MLB), with the aim to increase the superficial hydrophilicity of the system, allowing the diffusion trough the mucus layer. Once the complexation ability of the copolymer has been evaluated, obtaining nanosized polyplexes, polyplexes were functionalized on the surface with a thiolated TAT peptide, a cell-penetrating peptide (CPP), exploiting a thiol-ene reaction. TAT decorated polyplexes result to be highly cytocompatible and able to retain the siRNA with a suitable complexation weight ratio during the diffusion process through the mucus. Despite polyplexes establish weak bonds with the mucin chains, these can diffuse efficiently through the mucin layer and therefore potentially able to reach the bronchial epithelium. Furthermore, through cellular uptake studies, it was possible to observe how the obtained polyplexes penetrate effectively in the cytoplasm of bronchial epithelial cells, where they can reduce IL-8 gene expression, after LPS exposure. In the end, in order to obtain a formulation administrable as an inhalable dry powder, polyplexes were encapsulated in mannitol-based microparticles, by spray freeze drying, obtaining highly porous particles with proper technological characteristics that make them potentially administrable by inhalation route.
    Keywords:  Asthma; Polyamine; Polyaspartamide; Polyplexes; TAT; siRNA, STAT6
    DOI:  https://doi.org/10.1016/j.ejps.2023.106580
  7. Biomater Res. 2023 Sep 22. 27(1): 90
    mRNA nanodelivery systems: targeting strategies and administration routes.
    Mujie Yuan, Zeyu Han, Yan Liang, Yong Sun, Bin He, Wantao Chen, Fan Li.
      With the great success of coronavirus disease (COVID-19) messenger ribonucleic acid (mRNA) vaccines, mRNA therapeutics have gained significant momentum for the prevention and treatment of various refractory diseases. To function efficiently in vivo and overcome clinical limitations, mRNA demands safe and stable vectors and a reasonable administration route, bypassing multiple biological barriers and achieving organ-specific targeted delivery of mRNA. Nanoparticle (NP)-based delivery systems representing leading vector approaches ensure the successful intracellular delivery of mRNA to the target organ. In this review, chemical modifications of mRNA and various types of advanced mRNA NPs, including lipid NPs and polymers are summarized. The importance of passive targeting, especially endogenous targeting, and active targeting in mRNA nano-delivery is emphasized, and different cellular endocytic mechanisms are discussed. Most importantly, based on the above content and the physiological structure characteristics of various organs in vivo, the design strategies of mRNA NPs targeting different organs and cells are classified and discussed. Furthermore, the influence of administration routes on targeting design is highlighted. Finally, an outlook on the remaining challenges and future development toward mRNA targeted therapies and precision medicine is provided.
    Keywords:  Administration routes; Nanodelivery systems; Targeting; mRNA
    DOI:  https://doi.org/10.1186/s40824-023-00425-3
  8. J Appl Toxicol. 2023 Sep 18.
    Nonclinical safety assessment of an mRNA Covid-19 vaccine candidate following repeated administrations and biodistribution.
    Karine Broudic, Sébastien Laurent, Vladimir Perkov, Charlene Simon, Marie Garinot, Nathalie Truchot, Julie Latour, Paul Désert.
      Messenger RNA (mRNA) vaccines have demonstrated efficacy against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in humans. mRNA technology holds tremendous potential for rapid control and prevention of emergencies due to its flexibility with respect to production, application, and design for an efficacious and safe use in humans. We assessed the toxicity and biodistribution of MRT5500, an mRNA vaccine encoding for the full-length of the SARS-CoV-2 spike protein and delivered by lipid nanoparticles (LNPs) containing a novel ionizable lipid, Lipid-1 in preclinical animal models. In the repeated dose toxicity study, rabbits received three intramuscular (IM) injections of MRT5500 at 3-week interval followed by a 4-week observation period. In an exploratory biodistribution study in mice receiving a single IM injection of an mRNA encoding luciferase encapsulated in an LNP containing Lipid-1, the expression of the luciferase protein was monitored in vivo and ex vivo at several time points. In the regulatory biodistribution study in rabbits receiving a single IM injection of MRT5500, the quantification of the mRNA and the ionizable Lipid-1 were monitored in the same organs and time points as in the exploratory biodistribution study. MRT5500 was safe and well-tolerated with a transient acute phase response/inflammation and an expected vaccine-related immunological response, typical of those observed following a vaccine administration. The biodistribution data demonstrated that the mRNA and Lipid-1 components of the vaccine formulations were mainly detected at the injection site and in the draining lymph nodes. These results support the use of MRT5500 and its deployment into clinical trials.
    Keywords:  Covid-19; SARS-CoV-2; biodistribution; lipid nanoparticles; mRNA vaccine; repeated dose toxicity study
    DOI:  https://doi.org/10.1002/jat.4548
  9. J Chromatogr A. 2023 Sep 16. pii: S0021-9673(23)00600-3. [Epub ahead of print]1709 464375
    Optimization of HPLCCAD method for simultaneous analysis of different lipids in lipid nanoparticles with analytical QbD.
    Ki Hyun Kim, Ji Eun Lee, Jae Chul Lee, Ravi Maharjan, Hyunsuk Oh, Kyeong Lee, Nam Ah Kim, Seong Hoon Jeong.
      Since lipid nanoparticles (LNP) have emerged as a potent drug delivery system, the objective of this study was to develop and optimize a robust high-performance liquid chromatography with charged aerosol detectors (HPLCCAD) method to simultaneously quantify different lipids in LNPs using the analytical quality by design (AQbD) approach. After defining analytical target profile (ATP), critical method attributes (CMAs) were established as a resolution between the closely eluting lipid peaks and the total analysis time. Thus, potential high-risk method parameters were identified through the initial risk assessment. These parameters were screened using Plackett-Burman design, and three critical method parameters (CMPs)-MeOH ratio, flow rate, and column temperature-were selected for further optimization. Box-Behnken design was employed to develop the quadratic models that explain the relationship between the CMPs and CMAs and to determine the optimal operating conditions. Moreover, to ensure the robustness of the developed method, a method operable design region (MODR) was established using the Monte Carlo simulation. The MODR was identified within the probability map, where the risk of failure to achieve the desired CMAs was less than 1%. The optimized method was validated according to the ICH guidelines (linearity: R2 > 0.995, accuracy: 97.15-100.48% recovery, precision: RSD < 5%) and successfully applied for the analysis of the lipid in the LNP samples. The development of the analytical method to quantify the lipids is essential for the formulation development and quality control of LNP-based drugs since the potency of LNPs is significantly dependent on the compositions and contents of the lipids in the formation.
    Keywords:  Analytical quality by design; Charged aerosol detector; Design of experiment; Lipid nanoparticles
    DOI:  https://doi.org/10.1016/j.chroma.2023.464375
  10. Cell Death Dis. 2023 Sep 22. 14(9): 626
    In vivo self-assembly and delivery of VEGFR2 siRNA-encapsulated small extracellular vesicles for lung metastatic osteosarcoma therapy.
    Lingfeng Yu, Gentao Fan, Qingyan Wang, Yan Zhu, Hao Zhu, Jiang Chang, Zhen Wang, Shoubin Zhan, Xianming Hua, Diankun She, Jianhao Huang, Yicun Wang, Jianning Zhao, Chen-Yu Zhang, Xi Chen, Guangxin Zhou.
      The prognosis of lung metastatic osteosarcoma (OS) remains disappointing. siRNA-based gene silencing of VEGFR2 is a promising treatment strategy for lung metastatic OS, but there is a lack of safe and efficient delivery systems to encapsulate siRNAs for in vivo administration. This study presented a synthetic biological strategy that remolds the host liver with synthesized genetic circuits for efficient in vivo VEGFR2 siRNA delivery. After being taken-up by hepatocytes, the genetic circuit (in the form of a DNA plasmid) reprogrammed the liver to drive the autonomous intrahepatic assembly and encapsulation of VEGFR2 siRNAs into secretory small extracellular vesicles (sEVs), thus allowing for the transport of self-assembled VEGFR2 siRNAs towards the lung. The results showed that our strategy was superior to the positive medicine (Apatinib) for OS lung metastasis in terms of therapeutic efficacy and toxic adverse effects and may provide a feasible and viable therapeutic solution for lung metastatic OS.
    DOI:  https://doi.org/10.1038/s41419-023-06159-3
  11. Stem Cell Rev Rep. 2023 Sep 18.
    Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells.
    Malihe Lotfi, Dorsa Morshedi Rad, Samaneh Sharif Mashhadi, Atefeh Ashouri, Majid Mojarrad, Sina Mozaffari-Jovin, Shima Farrokhi, Maryam Hashemi, Marzieh Lotfi, Majid Ebrahimi Warkiani, Mohammad Reza Abbaszadegan.
      Rapid advancement in genome editing technologies has provided new promises for treating neoplasia, cardiovascular, neurodegenerative, and monogenic disorders. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful gene editing tool offering advantages, including high editing efficiency and low cost over the conventional approaches. Human pluripotent stem cells (hPSCs), with their great proliferation and differentiation potential into different cell types, have been exploited in stem cell-based therapy. The potential of hPSCs and the capabilities of CRISPR/Cas9 genome editing has been paradigm-shifting in medical genetics for over two decades. Since hPSCs are categorized as hard-to-transfect cells, there is a critical demand to develop an appropriate and effective approach for CRISPR/Cas9 delivery into these cells. This review focuses on various strategies for CRISPR/Cas9 delivery in stem cells.
    Keywords:  CRISPR/Cas9 system; Delivery technologies; Gene editing; Stem cell therapy; Stem cells
    DOI:  https://doi.org/10.1007/s12015-023-10585-3
  12. Adv Mater. 2023 Sep 21. e2306808
    Lipid-Centric Design of Plasma Membrane-Mimicking Nanocarriers for Targeted Chemotherapeutic Delivery.
    Di Ning, Zhi-Gang Wang, Lei Wang, Yi-Fan Tian, Fang Jing, Lin-Han Jiang, Meng-Qian Zhang, Yang-Yang Liu, Dai-Wen Pang, Wonhwa Cho, Shu-Lin Liu.
      The plasma membranes (PM) of mammalian cells contain diverse lipids, proteins and carbohydrates that are important for systemic recognition and communication in health and disease. Cell membrane coating technology that imparts unique properties of natural plasma membranes to the surface of encapsulated nanoparticles is thus becoming a powerful platform for drug delivery, immunomodulation, and vaccination. However, current coating methods fail to take full advantage of the natural systems because they disrupt the complex and functionally essential features of PMs, most notably the chemical diversity and compositional differences of lipids in two leaflets of the PM. Herein, we report a new lipid coating approach in which the lipid composition is optimized through a combination of biomimetic and systematic variation approaches for the custom design of nanocarrier systems for precision drug delivery. Nanocarriers coated with the optimized lipids offer unique advantages in terms of bioavailability and the efficiency in tumor targeting, tumor penetration, cellular uptake, and drug release. This pilot study provides new insight into the rational design and optimization of nanocarriers for cancer chemotherapeutic drugs, and lays the foundation for further customization of cell membrane-mimicking nanocarriers through systematic incorporation of other components. This article is protected by copyright. All rights reserved.
    Keywords:  biomimetic nanotechnology; chemotherapy; lipid, nanomedicine; plasma membrane
    DOI:  https://doi.org/10.1002/adma.202306808
  13. Expert Opin Drug Deliv. 2023 Sep 21.
    Inhalation delivery of nucleic acid gene therapies in preclinical drug development.
    Conor A Ruzycki, Derek Montoya, Hammad Irshad, Jason Cox, Yue Zhou, Jacob D McDonald, Philip J Kuehl.
       INTRODUCTION: Inhaled gene therapy programs targeting diseases of the lung have seen increasing interest in recent years, though as of yet no product has successfully entered the market. Preclinical research to support such programs is critically important in maximizing the chances of developing successful candidates.
    AREAS COVERED: Aspects of inhalation delivery of gene therapies are reviewed, with a focus on preclinical research in animal models. Various barriers to inhalation delivery of gene therapies are discussed, including aerosolization stresses, aerosol behavior in the respiratory tract, and disposition processes post-deposition. Important aspects of animal models are considered, including determinations of biologically-relevant determinations of dose and issues related to translatability.
    EXPERT OPINION: Development of clinically-efficacious inhaled gene therapies has proven difficult owing to numerous challenges. Fit-for-purpose experimental and analytical methods are necessary for determinations of biologically-relevant doses in preclinical animal models. Further developments in disease-specific animal models may aid in improving translatability of results in future work, and we expect to see accelerated interests in inhalation gene therapies for various diseases. Sponsors, researchers, and regulators are encouraged to engage in early and frequent discussion regarding candidate therapies, and additional dissemination of preclinical methodologies would be of immense value in avoiding common pitfalls.
    Keywords:  Alpha-1 antitrypsin deficiency; Asthma; Cystic fibrosis; Dose determination; Inhaled gene therapies; Lung cancer; animal models; preclinical drug development; translatability
    DOI:  https://doi.org/10.1080/17425247.2023.2261369
  14. Signal Transduct Target Ther. 2023 Sep 20. 8(1): 365
    mRNA vaccines in disease prevention and treatment.
    Gang Zhang, Tianyu Tang, Yinfeng Chen, Xing Huang, Tingbo Liang.
      mRNA vaccines have emerged as highly effective strategies in the prophylaxis and treatment of diseases, thanks largely although not totally to their extraordinary performance in recent years against the worldwide plague COVID-19. The huge superiority of mRNA vaccines regarding their efficacy, safety, and large-scale manufacture encourages pharmaceutical industries and biotechnology companies to expand their application to a diverse array of diseases, despite the nonnegligible problems in design, fabrication, and mode of administration. This review delves into the technical underpinnings of mRNA vaccines, covering mRNA design, synthesis, delivery, and adjuvant technologies. Moreover, this review presents a systematic retrospective analysis in a logical and well-organized manner, shedding light on representative mRNA vaccines employed in various diseases. The scope extends across infectious diseases, cancers, immunological diseases, tissue damages, and rare diseases, showcasing the versatility and potential of mRNA vaccines in diverse therapeutic areas. Furthermore, this review engages in a prospective discussion regarding the current challenge and potential direction for the advancement and utilization of mRNA vaccines. Overall, this comprehensive review serves as a valuable resource for researchers, clinicians, and industry professionals, providing a comprehensive understanding of the technical aspects, historical context, and future prospects of mRNA vaccines in the fight against various diseases.
    DOI:  https://doi.org/10.1038/s41392-023-01579-1
  15. J Extracell Vesicles. 2023 09;12(9): e12343
    Engineering extracellular vesicles to deliver CRISPR ribonucleoprotein for gene editing.
    Joseph Andrew Whitley, Houjian Cai.
      Clustered regularly interspaced palindromic repeats (CRISPR) is a gene editing tool with tremendous therapeutic potential. Recently, ribonucleoprotein (RNP) complex-based CRISPR systems have gained momentum due to their reduction of off-target editing. This has coincided with the emergence of extracellular vesicles (EVs) as a therapeutic delivery vehicle due to its low immunogenicity and high capacity for manipulation. EVs are cell-derived membranous nanoparticles which mediate the intercellular transfer of molecular components. Current technologies achieve CRISPR RNP encapsulation into EVs through EVs biogenesis, thereby avoiding unnecessary physical, chemical or biological manipulations to the vesicles directly. Herein, we identify sixteen EVs-based CRISPR RNP encapsulation strategies, each with distinct genetic features to encapsulate CRISPR RNP. According to the molecular mechanism facilitating the encapsulation process, there are six strategies of encapsulating Cas9 RNP into virus-like particles based on genetic fusion, seven into EVs based on protein tethering, and three based on sgRNA-coupled encapsulation. Additionally, the incorporation of a targeting moiety to the EVs membrane surface through EVs biogenesis confers tropism and increases delivery efficiency to specific cell types. The targeting moieties include viral envelope proteins, recombinant proteins containing a ligand peptide, single-chain fragment variable (scFv) antibodies, and integrins. However, current strategies still have a number of limitations which prevent their use in clinical trials. Among those, the incorporation of viral proteins for encapsulation of Cas9 RNP have raised issues of biocompatibility due to host immune response. Future studies should focus on genetically engineering the EVs without viral proteins, enhancing EVs delivery specificity, and promoting EVs-based homology directed repair. Nevertheless, the integration of CRISPR RNP encapsulation and tropism technologies will provide strategies for the EVs-based delivery of CRISPR RNP in gene therapy and disease treatment.
    Keywords:  CRISPR; Cas9/sgRNA ribonucleoprotein; delivery; encapsulation; extracellular vesicles; tropism
    DOI:  https://doi.org/10.1002/jev2.12343
  16. Nat Commun. 2023 Sep 21. 14(1): 5663
    mRNA vaccine quality analysis using RNA sequencing.
    Helen M Gunter, Senel Idrisoglu, Swati Singh, Dae Jong Han, Emily Ariens, Jonathan R Peters, Ted Wong, Seth W Cheetham, Jun Xu, Subash Kumar Rai, Robert Feldman, Andy Herbert, Esteban Marcellin, Romain Tropee, Trent Munro, Tim R Mercer.
      The success of mRNA vaccines has been realised, in part, by advances in manufacturing that enabled billions of doses to be produced at sufficient quality and safety. However, mRNA vaccines must be rigorously analysed to measure their integrity and detect contaminants that reduce their effectiveness and induce side-effects. Currently, mRNA vaccines and therapies are analysed using a range of time-consuming and costly methods. Here we describe a streamlined method to analyse mRNA vaccines and therapies using long-read nanopore sequencing. Compared to other industry-standard techniques, VAX-seq can comprehensively measure key mRNA vaccine quality attributes, including sequence, length, integrity, and purity. We also show how direct RNA sequencing can analyse mRNA chemistry, including the detection of nucleoside modifications. To support this approach, we provide supporting software to automatically report on mRNA and plasmid template quality and integrity. Given these advantages, we anticipate that RNA sequencing methods, such as VAX-seq, will become central to the development and manufacture of mRNA drugs.
    DOI:  https://doi.org/10.1038/s41467-023-41354-y
  17. Nanoscale Horiz. 2023 Sep 19.
    Microfluidic synthesis of nanomaterials for biomedical applications.
    Yanjuan Huang, Chao Liu, Qiang Feng, Jiashu Sun.
      The field of nanomaterials has progressed dramatically over the past decades with important contributions to the biomedical area. The physicochemical properties of nanomaterials, such as the size and structure, can be controlled through manipulation of mass and heat transfer conditions during synthesis. In particular, microfluidic systems with rapid mixing and precise fluid control are ideal platforms for creating appropriate synthesis conditions. One notable example of microfluidics-based synthesis is the development of lipid nanoparticle (LNP)-based mRNA vaccines with accelerated clinical translation and robust efficacy during the COVID-19 pandemic. In addition to LNPs, microfluidic systems have been adopted for the controlled synthesis of a broad range of nanomaterials. In this review, we introduce the fundamental principles of microfluidic technologies including flow field- and multiple field-based methods for fabricating nanoparticles, and discuss their applications in the biomedical field. We conclude this review by outlining several major challenges and future directions in the implementation of microfluidic synthesis of nanomaterials.
    DOI:  https://doi.org/10.1039/d3nh00217a
  18. Sci Adv. 2023 Sep 22. 9(38): eadh1655
    Intranasal mRNA-LNP vaccination protects hamsters from SARS-CoV-2 infection.
    Gabriela Baldeon Vaca, Michelle Meyer, Ana Cadete, Chiaowen Joyce Hsiao, Anne Golding, Albert Jeon, Eric Jacquinet, Emily Azcue, Chenxia Monica Guan, Xavier Sanchez-Felix, Colette A Pietzsch, Chad E Mire, Matthew A Hyde, Margaret E Comeaux, Julie M Williams, Jean C Sung, Andrea Carfi, Darin K Edwards, Alexander Bukreyev, Kapil Bahl.
      Intranasal vaccination represents a promising approach for preventing disease caused by respiratory pathogens by eliciting a mucosal immune response in the respiratory tract that may act as an early barrier to infection and transmission. This study investigated immunogenicity and protective efficacy of intranasally administered messenger RNA (mRNA)-lipid nanoparticle (LNP) encapsulated vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Syrian golden hamsters. Intranasal mRNA-LNP vaccination systemically induced spike-specific binding [immunoglobulin G (IgG) and IgA] and neutralizing antibodies. Intranasally vaccinated hamsters also had decreased viral loads in the respiratory tract, reduced lung pathology, and prevented weight loss after SARS-CoV-2 challenge. Together, this study demonstrates successful immunogenicity and protection against respiratory viral infection by an intranasally administered mRNA-LNP vaccine.
    DOI:  https://doi.org/10.1126/sciadv.adh1655
  19. EMBO J. 2023 Sep 20. e114760
    RNA-based medicine: from molecular mechanisms to therapy.
    Anke Sparmann, Jörg Vogel.
      RNA-based therapeutics have the potential to revolutionize the treatment and prevention of human diseases. While early research faced setbacks, it established the basis for breakthroughs in RNA-based drug design that culminated in the extraordinarily fast development of mRNA vaccines to combat the COVID-19 pandemic. We have now reached a pivotal moment where RNA medicines are poised to make a broad impact in the clinic. In this review, we present an overview of different RNA-based strategies to generate novel therapeutics, including antisense and RNAi-based mechanisms, mRNA-based approaches, and CRISPR-Cas-mediated genome editing. Using three rare genetic diseases as examples, we highlight the opportunities, but also the challenges to wide-ranging applications of this class of drugs.
    Keywords:  CRISPR-Cas therapy; RNA interference; antisense oligonucleotide drug; mRNA therapeutics; rare genetic disease
    DOI:  https://doi.org/10.15252/embj.2023114760
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