bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2022‒08‒28
twelve papers selected by
the Merkel lab
Ludwig-Maximilians University
  1. Spheroplexes: Hybrid PLGA-cationic lipid nanoparticles, for in vitro and oral delivery of siRNA.
  2. Efficient Delivery of DNA Using Lipid Nanoparticles.
  3. mRNA-Loaded Lipid Nanoparticles Targeting Immune Cells in the Spleen for Use as Cancer Vaccines.
  4. mRNA-Loaded Lipid Nanoparticles Targeting Dendritic Cells for Cancer Immunotherapy.
  5. Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo.
  6. Two quality and stability indicating imaged CIEF methods for mRNA vaccines.
  7. Monitoring Anti-PEG Antibodies Level upon Repeated Lipid Nanoparticle-Based COVID-19 Vaccine Administration.
  8. Vutrisiran: First Approval.
  9. Polyglycerol fatty acid esters as alternatives to PEGylated lipids for liposome coating.
  10. A microfluidic system for rapid nucleic acid analysis based on real-time convective PCR at point-of-care testing.
  11. Rapid Extraction of Viral Nucleic Acids Using Rotating Blade Lysis and Magnetic Beads.
  12. Feasibility and application of machine learning enabled fast screening of poly-beta-amino-esters for cartilage therapies.
  1. J Control Release. 2022 Aug 19. pii: S0168-3659(22)00535-1. [Epub ahead of print]
    Spheroplexes: Hybrid PLGA-cationic lipid nanoparticles, for in vitro and oral delivery of siRNA.
    Danielle Campiol Arruda, Anne-Marie Lachagès, Hélène Demory, Guillaume Escriou, René Lai-Kuen, Pierre-Yves Dugas, Céline Hoffmann, Stéphanie Bessoles, Guillaume Sarrabayrouse, Angelo Malachias, Stéphanie Finet, Pedro Lana Gastelois, Waldemar Augusto de Almeida Macedo, Armando da Silva Cunha, Pascal Bigey, Virginie Escriou.
      Vectorized small interfering RNAs (siRNAs) are widely used to induce gene silencing. Among the delivery systems used, lipid-based particles are the most effective. Our objective was the development of novel lipid-polymer hybrid nanoparticles, from lipoplexes (complexes of cationic lipid and siRNAs), and poly (lactic-co-glycolic acid) (PLGA), using a simple modified nanoprecipitation method. Due to their morphology, we called these hybrid nanoparticles Spheroplexes. We elucidated their structure using several physico-chemical techniques and showed that they are composed of a hydrophobic PLGA matrix, surrounded by a lipid envelope adopting a lamellar structure, in which the siRNA is complexed, and they retain surface characteristics identical to the starting nanoparticles, i.e. lipoplexes siRNA. We analyzed the composition of the particle population and determined the final percentage of spheroplexes within this population, 80 to 85% depending on the preparation conditions, using fluorescent markers and the ability of flow cytometry to detect nanometric particles (approximately 200 nm). Finally, we showed that spheroplexes are very stable particles and more efficient than siRNA lipoplexes for the delivery of siRNA to cultured cells. We administered spheroplexes contain siRNAs targeting TNF-α to mice with ulcerative colitis induced by dextran sulfate and our results indicate a disease regression effect with a response probably mediated by their uptake by macrophages / monocytes at the level of lamina propria of the colon. The efficacy of decreased level of TNF-α in vivo seemed to be an association of spheroplexes polymer-lipid composition and the specific siRNA. These results demonstrate that spheroplexes are a promising hybrid nanoparticle for the oral delivery of siRNA to the colon.
    Keywords:  Biodegradable polymer; Delivery system; Hybrid nanoparticle; Lipoplexes; Oral delivery; RNA interference
    DOI:  https://doi.org/10.1016/j.jconrel.2022.08.030
  2. Pharmaceutics. 2022 Aug 15. pii: 1698. [Epub ahead of print]14(8):
    Efficient Delivery of DNA Using Lipid Nanoparticles.
    Lishan Cui, Serena Renzi, Erica Quagliarini, Luca Digiacomo, Heinz Amenitsch, Laura Masuelli, Roberto Bei, Gianmarco Ferri, Francesco Cardarelli, Junbiao Wang, Augusto Amici, Daniela Pozzi, Cristina Marchini, Giulio Caracciolo.
      DNA vaccination has been extensively studied as a promising strategy for tumor treatment. Despite the efforts, the therapeutic efficacy of DNA vaccines has been limited by their intrinsic poor cellular internalization. Electroporation, which is based on the application of a controlled electric field to enhance DNA penetration into cells, has been the method of choice to produce acceptable levels of gene transfer in vivo. However, this method may cause cell damage or rupture, non-specific targeting, and even degradation of pDNA. Skin irritation, muscle contractions, pain, alterations in skin structure, and irreversible cell damage have been frequently reported. To overcome these limitations, in this work, we use a microfluidic platform to generate DNA-loaded lipid nanoparticles (LNPs) which are then characterized by a combination of dynamic light scattering (DLS), synchrotron small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). Despite the clinical successes obtained by LNPs for mRNA and siRNA delivery, little is known about LNPs encapsulating bulkier DNA molecules, the clinical application of which remains challenging. For in vitro screening, LNPs were administered to human embryonic kidney 293 (HEK-293) and Chinese hamster ovary (CHO) cell lines and ranked for their transfection efficiency (TE) and cytotoxicity. The LNP formulation exhibiting the highest TE and the lowest cytotoxicity was then tested for the delivery of the DNA vaccine pVAX-hECTM targeting the human neoantigen HER2, an oncoprotein overexpressed in several cancer types. Using fluorescence-activated cell sorting (FACS), immunofluorescence assays and fluorescence confocal microscopy (FCS), we proved that pVAX-hECTM-loaded LNPs produce massive expression of the HER2 antigen on the cell membrane of HEK-293 cells. Our results provide new insights into the structure-activity relationship of DNA-loaded LNPs and pave the way for the access of this gene delivery technology to preclinical studies.
    Keywords:  DNA vaccines; HER2; lipid nanoparticles
    DOI:  https://doi.org/10.3390/pharmaceutics14081698
  3. Pharmaceuticals (Basel). 2022 Aug 18. pii: 1017. [Epub ahead of print]15(8):
    mRNA-Loaded Lipid Nanoparticles Targeting Immune Cells in the Spleen for Use as Cancer Vaccines.
    Ryoya Shimosakai, Ikramy A Khalil, Seigo Kimura, Hideyoshi Harashima.
      mRNA delivery has recently gained substantial interest for possible use in vaccines. Recently approved mRNA vaccines are administered intramuscularly where they transfect antigen-presenting cells (APCs) near the site of administration, resulting in an immune response. The spleen contains high numbers of APCs, which are located near B and T lymphocytes. Therefore, transfecting APCs in the spleen would be expected to produce a more efficient immune response, but this is a challenging task due to the different biological barriers. Success requires the development of an efficient system that can transfect different immune cells in the spleen. In this study, we report on the development of mRNA-loaded lipid nanoparticles (LNPs) targeting immune cells in the spleen with the goal of eliciting an efficient immune response against the antigen encoded in the mRNA. The developed system is composed of mRNA loaded in LNPs whose lipid composition was optimized for maximum transfection into spleen cells. Dendritic cells, macrophages and B cells in the spleen were efficiently transfected. The optimized LNPs produced efficient dose-dependent cytotoxic T lymphocyte activities that were significantly higher than that produced after local administration. The optimized LNPs encapsulating tumor-antigen encoding mRNA showed both prophylactic and therapeutic antitumor effects in mice.
    Keywords:  immune cell; lipid nanoparticle; mRNA; spleen; vaccine
    DOI:  https://doi.org/10.3390/ph15081017
  4. Pharmaceutics. 2022 Jul 28. pii: 1572. [Epub ahead of print]14(8):
    mRNA-Loaded Lipid Nanoparticles Targeting Dendritic Cells for Cancer Immunotherapy.
    Kosuke Sasaki, Yusuke Sato, Kento Okuda, Kazuki Iwakawa, Hideyoshi Harashima.
      Dendritic cells (DCs) are attractive antigen-presenting cells to be targeted for vaccinations. However, the systemic delivery of mRNA to DCs is hampered by technical challenges. We recently reported that it is possible to regulate the size of RNA-loaded lipid nanoparticles (LNPs) to over 200 nm with the addition of salt during their formation when a microfluidic device is used and that larger LNPs delivered RNA more efficiently and in greater numbers to splenic DCs compared to the smaller counterparts. In this study, we report on the in vivo optimization of mRNA-loaded LNPs for use in vaccines. The screening included a wide range of methods for controlling particle size in addition to the selection of an appropriate lipid type and its composition. The results showed a clear correlation between particle size, uptake and gene expression activity in splenic DCs and indicated that a size range from 200 to 500 nm is appropriate for use in targeting splenic DCs. It was also found that it was difficult to predict the transgene expression activity and the potency of mRNA vaccines in splenic DCs using the whole spleen. A-11-LNP, which was found to be the optimal formulation, induced better transgene expression activity and maturation in DCs and induced clear therapeutic antitumor effects in an E.G7-OVA tumor model compared to two clinically relevant LNP formulations.
    Keywords:  cancer immunotherapy; delivery; dendritic cells; lipid nanoparticles; mRNA; particle size
    DOI:  https://doi.org/10.3390/pharmaceutics14081572
  5. J Control Release. 2022 Aug 21. pii: S0168-3659(22)00538-7. [Epub ahead of print]
    Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo.
    Jing-E Zhou, Lei Sun, Yujie Jia, Zhehao Wang, Tengshuo Luo, Jingwen Tan, Xiaoyan Fang, Hongjia Zhu, Jing Wang, Lei Yu, Zhiqiang Yan.
      Chimeric receptor T cells (CAR-T) can effectively cure leukemia; however, there are two limitations: a complicated preparation process ex vivo and cytokine release syndrome (CRS). In this study, we constructed a lipid nanoparticle system modified by CD3 antibody on the surface, loading with the plasmid containing the combination gene of interleukin 6 short hairpin RNA (IL-6 shRNA) and CD19-CAR (AntiCD3-LNP/CAR19 + shIL6). The system targeted T cells by the mediation of CD3 antibody and stably transfected T cells to transform them into CAR-T cells with IL-6 knockdown, thus killing CD19-highly expressed leukemia tumor cells and reducing CRS caused by IL-6. In vivo experiments showed that AntiCD3-LNP/CAR19 + shIL6 could stably transfect T cells and produce CAR-T within 90 days to kill the tumor. This significantly prolonged the survival time of leukemia model mice and demonstrated the prepared LNP exhibited the same anti-tumor effect as the traditional CAR-T cells prepared ex vivo. In this study, CAR-T cells were directly produced in vivo after intravenous injection of the lipid nanoparticles, without the need of using the current complex process ex vivo. Additionally, IL-6 expression was silenced, which would be helpful to reduce the CRS and improve the safety of CAR-T therapy. This method improves the convenience of using CAR-T technology and is helpful in further promoting the clinical application of CAR-T.
    Keywords:  CD3 antibody; Chimeric antigen receptor T cells; Cytokine release syndrome; IL-6 shRNA; Lipid nanoparticles
    DOI:  https://doi.org/10.1016/j.jconrel.2022.08.033
  6. Electrophoresis. 2022 Aug 24.
    Two quality and stability indicating imaged CIEF methods for mRNA vaccines.
    Finja Krebs, Udo Burger, Susanne Dörks, Markus Kramer, Hermann Wätzig.
      Two imaged CIEF methods were developed to provide insight into the quality and stability of messenger ribonucleic acid (mRNA) vaccines, specifically, mRNA encapsulated in lipid nanoparticles (LNPs). A variety of stressed and lipid composition-modified samples were measured and detected by their UV absorption. The results were supported by the data of an encapsulation assay and particle sizing. One method, using 9 M urea as an additive, shows two broad and jagged peaks in which the peak shape offers detailed information. The summed peak area of both peaks showed RSDs from 2 to 8% when one batch was measured in triplicate and apparently depends on the size of the LNPs. In the second method, a combination of 5.5 M urea and 2 M N-ethylurea was used. This method is characterized by high repeatability of the pI value (< 0.5%). The repeatable peak area (RSD of 2-7%) correlates linearly with the mRNA content, which also applies to the first method, and added stress is evident by the change in pI and peak area. Furthermore, experiments with the addition of a fluorescent dye were performed (fluorescence detection), which tremendously increased the sensitivity of the methods. Both methods can be used to characterize the stability of mRNA-loaded LNPs, for example when investigating various storage times at different temperatures and freeze-thaw cycles, as well as the ability of the methods to distinguish lipid compositions and measure batch-to-batch variability. This article is protected by copyright. All rights reserved.
    Keywords:  CIEF; lipid nanoparticles; mRNA; method development; vaccines
    DOI:  https://doi.org/10.1002/elps.202200123
  7. Int J Mol Sci. 2022 Aug 09. pii: 8838. [Epub ahead of print]23(16):
    Monitoring Anti-PEG Antibodies Level upon Repeated Lipid Nanoparticle-Based COVID-19 Vaccine Administration.
    Giuditta Guerrini, Sabrina Gioria, Aisha V Sauer, Simone Lucchesi, Francesca Montagnani, Gabiria Pastore, Annalisa Ciabattini, Donata Medaglini, Luigi Calzolai.
      PEGylated lipids are one of the four constituents of lipid nanoparticle mRNA COVID-19 vaccines. Therefore, various concerns have been raised on the generation of anti-PEG antibodies and their potential role in inducing hypersensitivity reactions following vaccination or in reducing vaccine efficacy due to anti-carrier immunity. Here, we assess the prevalence of anti-PEG antibodies, in a cohort of vaccinated individuals, and give an overview of their time evolution after repeated vaccine administrations. Results indicate that, in our cohort, the presence of PEG in the formulation did not influence the level of anti-Spike antibodies generated upon vaccination and was not related to any reported, serious adverse effects. The time-course analysis of anti-PEG IgG showed no significant booster effect after each dose, whereas for IgM a significant increase in antibody levels was detected after the first and third dose. Data suggest that the presence of PEG in the formulation does not affect safety or efficacy of lipid-nanoparticle-based COVID-19 vaccines.
    Keywords:  COVID-19; SARS-CoV-2; anti-PEG Ig; anti-Spike Ig; lipid-nanoparticle-mRNA (LNP-mRNA)-based vaccine
    DOI:  https://doi.org/10.3390/ijms23168838
  8. Drugs. 2022 Aug 23.
    Vutrisiran: First Approval.
    Susan J Keam.
      Vutrisiran (AMVUTTRA™) is a subcutaneously administered transthyretin-directed small interfering ribonucleic acid (siRNA) therapeutic (also called RNA interference, or RNAi therapeutic) being developed by Alnylam Pharmaceuticals, Inc. for the treatment of amyloid transthyretin-mediated (ATTR) amyloidosis, including hereditary ATTR (hATTR) amyloidosis and wild-type ATTR (wtATTR) amyloidosis. Vutrisiran was approved in June 2022 in the USA for the treatment of the polyneuropathy of hATTR amyloidosis in adults and received a positive opinion in the EU in July 2022 for the treatment of hATTR amyloidosis in adult patients with stage 1 or stage 2 polyneuropathy. Vutrisiran is also under regulatory review for the treatment of the polyneuropathy of hATTR amyloidosis in adults in Japan and Brazil. This article summarizes the milestones in the development of vutrisiran leading to this first approval in hATTR amyloidosis.
    DOI:  https://doi.org/10.1007/s40265-022-01765-5
  9. Nanomedicine (Lond). 2022 Aug 24.
    Polyglycerol fatty acid esters as alternatives to PEGylated lipids for liposome coating.
    Huali Chen, Qianyu Zhang.
      Background: Polyglycerol (PG) is a type of biocompatible hydrophilic polyether polyol, and it is considered as a potential alternative to polyethylene glycol (PEG) in modifying nanomedicines. Materials & methods: Polyglycerol fatty acid esters (PGFEs) were modified onto liposomes and their serum stability, pharmacokinetics, in vivo distribution and the capacity to induce anti-PEG IgM were compared with PEGylated liposomes (PEG-Lips). Results: Polyglycerol 10-monostearate (PG-10-MS) displayed considerable serum stability and compatibility with mice red blood cells, and it significantly prolonged the blood circulation of liposomes in the pharmacokinetics study compared with the unmodified liposomes, with a similar biodistribution pattern to that of the PEG-Lips. Moreover, PGFE-modified liposomes were less likely to induce the production of anti-PEG IgM. Conclusion: PGFEs could be considered as good candidates to replace PEG lipids for the preparation of liposomes.
    Keywords:  PEGylation; anti-PEG IgM; liposomes; pharmacokinetics; polyglycerol fatty acid esters
    DOI:  https://doi.org/10.2217/nnm-2022-0101
  10. Microfluid Nanofluidics. 2022 ;26(9): 69
    A microfluidic system for rapid nucleic acid analysis based on real-time convective PCR at point-of-care testing.
    Donglin Xu, Xiaodan Jiang, Tianli Zou, Guijun Miao, Qiang Fu, Fei Xiang, Liang Feng, Xiangzhong Ye, Lulu Zhang, Xianbo Qiu.
      A microfluidic system for rapid nucleic acid analysis based on real-time convective PCR is developed. To perform 'sample-in, answer-out' nucleic acid analysis, a microfluidic chip is developed to efficiently extract nucleic acid, and meanwhile convective PCR (CPCR) is applied for rapid nucleic acid amplification. With an integrated microfluidic chip consisting of reagent pre-storage chambers, a lysis & wash chamber, an elution chamber and a waste chamber, nucleic acid extraction based on magnetic beads can be automatically performed for a large size of test sample within a limited time. Based on an easy-to-operate strategy, different pre-stored reagents can be conveniently released for consecutive reaction at different steps. To achieve efficient mixing, a portable companion device is developed to introduce properly controlled 3-D actuation to magnetic beads in nucleic acid extraction. In CPCR amplification, PCR reagent can be spontaneously and repeatedly circulated between hot and cool zones of the reactor for space-domain thermal cycling based on pseudo-isothermal heating. A handheld real-time CPCR device is developed to perform nucleic acid amplification and in-situ detection. To extend the detection throughput, multiple handheld real-time CPCR devices can be grouped together by a common control system. It is demonstrated that influenza A (H1N1) viruses with the reasonable concentration down to 1.0 TCID50/ml can be successfully detected with the microfluidic system.
    Keywords:  Convective PCR; Microfluidic chip; Mixing; Nucleic acid analysis; Nucleic acid extraction; Point-of-care (POC) testing
    DOI:  https://doi.org/10.1007/s10404-022-02577-5
  11. Diagnostics (Basel). 2022 Aug 17. pii: 1995. [Epub ahead of print]12(8):
    Rapid Extraction of Viral Nucleic Acids Using Rotating Blade Lysis and Magnetic Beads.
    Minju Bae, Junsoo Park, Hyeonah Seong, Hansol Lee, Wonsuk Choi, Jiyun Noh, Woojoo Kim, Sehyun Shin.
      The complex and lengthy protocol of current viral nucleic acid extraction processes limits their use outside laboratory settings. Here, we describe a rapid and reliable method for extracting nucleic acids from viral samples using a rotating blade and magnetic beads. The viral membrane can be instantly lysed using a high-speed rotating blade, and nucleic acids can be immediately isolated using a silica magnetic surface. The process was completed within 60 s by this method. Routine washing and eluting processes were subsequently conducted within 5 min. The results achieved by this method were comparable to those of a commercially available method. When the blade-based lysis and magnetic bead adsorption processes were performed separately, the RNA recovery rate was very low, and the Ct value was delayed compared to simultaneous lysis and RNA adsorption. Overall, this method not only dramatically shortens the conventional extraction time but also allows for its convenient use outside the laboratory, such as at remote field sites and for point-of-care testing.
    Keywords:  blade; extraction; lysis; magnetic beads; nucleic acid; virus
    DOI:  https://doi.org/10.3390/diagnostics12081995
  12. Sci Rep. 2022 Aug 20. 12(1): 14215
    Feasibility and application of machine learning enabled fast screening of poly-beta-amino-esters for cartilage therapies.
    Stefano Perni, Polina Prokopovich.
      Despite the large prevalence of diseases affecting cartilage (e.g. knee osteoarthritis affecting 16% of population globally), no curative treatments are available because of the limited capacity of drugs to localise in such tissue caused by low vascularisation and electrostatic repulsion. While an effective delivery system is sought, the only option is using high drug doses that can lead to systemic side effects. We introduced poly-beta-amino-esters (PBAEs) to effectively deliver drugs into cartilage tissues. PBAEs are copolymer of amines and di-acrylates further end-capped with other amine; therefore encompassing a very large research space for the identification of optimal candidates. In order to accelerate the screening of all possible PBAEs, the results of a small pool of polymers (n = 90) were used to train a variety of machine learning (ML) methods using only polymers properties available in public libraries or estimated from the chemical structure. Bagged multivariate adaptive regression splines (MARS) returned the best predictive performance and was used on the remaining (n = 3915) possible PBAEs resulting in the recognition of pivotal features; a further round of screening was carried out on PBAEs (n = 150) with small variations of structure of the main candidates from the first round. The refinements of such characteristics enabled the identification of a leading candidate predicted to improve drug uptake > 20 folds over conventional clinical treatment; this uptake improvement was also experimentally confirmed. This work highlights the potential of ML to accelerate biomaterials development by efficiently extracting information from a limited experimental dataset thus allowing patients to benefit earlier from a new technology and at a lower price. Such roadmap could also be applied for other drug/materials development where optimisation would normally be approached through combinatorial chemistry.
    DOI:  https://doi.org/10.1038/s41598-022-18332-3
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