bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2023–04–02
twelve papers selected by
the Merkel lab, Ludwig-Maximilians University
  1. Delivery of siRNA using hyaluronic acid-guided nanoparticles for downregulation of CXCR4.
  2. Influence of lipid composition of messenger RNA-loaded lipid nanoparticles on the protein expression via intratracheal administration in mice.
  3. The Development of Cell-Penetrating Peptides for Efficient and Selective In Vivo Expression of mRNA in Spleen Tissue.
  4. A New Optimization Strategy of Highly Branched Poly(β-Amino Ester) for Enhanced Gene Delivery: Removal of Small Molecular Weight Components.
  5. Lysine-Derived Charge-Altering Releasable Transporters: Targeted Delivery of mRNA and siRNA to the Lungs.
  6. Molecular Chameleon Carriers for Nucleic Acid Delivery: The Sweet Spot Between Lipoplexes and Polyplexes.
  7. A New Strategy for Nucleic Acid Delivery and Protein Expression Using Biocompatible Nanohydrogels of Predefined Sizes.
  8. Nanoparticles (NPs)-mediated systemic mRNA delivery to reverse trastuzumab resistance for effective breast cancer therapy.
  9. Sodium alginate coating simultaneously increases the biosafety and immunotherapeutic activity of the cationic mRNA nanovaccine.
  10. Combinatorial design of nanoparticles for pulmonary mRNA delivery and genome editing.
  11. Mechanism-Driven Technology Development for Solving the Intracellular Delivery Problem of Hard-To-Transfect Cells.
  12. Plant-Derived Extracellular Vesicles as a Delivery Platform for RNA-Based Vaccine: Feasibility Study of an Oral and Intranasal SARS-CoV-2 Vaccine.
  1. Biopolymers. 2023 Mar 27. e23535
    Delivery of siRNA using hyaluronic acid-guided nanoparticles for downregulation of CXCR4.
    Beste Çağdaş Tunalı, Eda Çelik, Fatma Azize Budak Yıldıran, Mustafa Türk.
      In this study, effective transport of small interfering RNAs (siRNAs) via hyaluronic acid (HA) receptor was carried out with biodegradable HA and low-molecular weight polyethyleneimine (PEI)-based transport systems. Gold nanoparticles (AuNPs) capable of giving photothermal response, and their conjugates with PEI and HA, were also added to the structure. Thus, a combination of gene silencing, photothermal therapy and chemotherapy, has been accomplished. The synthesized transport systems ranged in size, between 25 and 690 nm. When the particles were applied at a concentration of 100 μg mL-1 (except AuPEI NPs) in vitro, cell viability was above 50%. Applying radiation after the conjugate/siRNA complex (especially those containing AuNP) treatment, increased the cytotoxic effect (decrease in cell viability of 37%, 54%, 13%, and 15% for AuNP, AuPEI NP, AuPEI-HA, and AuPEI-HA-DOX, respectively) on the MDA-MB-231 cell line. CXCR4 gene silencing via the synthesized complexes, especially AuPEI-HA-DOX/siRNA was more efficient in MDA-MB-231 cells (25-fold decrease in gene expression) than in CAPAN-1 cells. All these results demonstrated that the synthesized PEI-HA and AuPEI-HA-DOX conjugates can be used as siRNA carriers that are particularly effective, especially in the treatment of breast cancer.
    Keywords:  CXCR4; Polyethylenimine; gene therapy; gold nanoparticle; hyaluronic acid; siRNA
    DOI:  https://doi.org/10.1002/bip.23535
  2. Int J Pharm. 2023 Mar 25. pii: S0378-5173(23)00316-2. [Epub ahead of print] 122896
    Influence of lipid composition of messenger RNA-loaded lipid nanoparticles on the protein expression via intratracheal administration in mice.
    Longjian Geng, Naoya Kato, Yukinobu Kodama, Hidefumi Mukai, Shigeru Kawakami.
      Intratracheal (i.t.) administration, which takes advantage of the specific structure of the respiratory system, can effectively deliver nanoparticles to the lung. Much remains unknown about the i.t. administration of messenger RNA (mRNA)-lipid nanoparticles (LNPs) and the effect of lipid composition. In this study, we administered minute amounts of mRNA-LNP solutions into mice intratracheally and investigated the effect of lipid composition on protein expression in the lungs. We first validated higher protein expression with mRNA-LNP compared to that with mRNA-PEI complex and naked mRNA. Then, we evaluated the influence of lipid composition of LNPs on the protein expression and found that: 1) decreasing the PEG molarity from 1.5% to 0.5% could significantly increase the protein expression; 2) replacing DMG-PEG with DSG-PEG could slightly increase the protein expression; 3) using DOPE instead of DSPC could increase protein expression by an order of magnitude. We successfully prepared an mRNA-LNP with optimal lipid compositions that led to robust protein expression following i.t. administration, thus providing meaningful insights into advanced development of mRNA-LNPs for therapeutic i.t. administration.
    Keywords:  Intratracheal administration; Lipid composition; Lipid nanoparticle; Lung; Protein expression
    DOI:  https://doi.org/10.1016/j.ijpharm.2023.122896
  3. Pharmaceutics. 2023 Mar 15. pii: 952. [Epub ahead of print]15(3):
    The Development of Cell-Penetrating Peptides for Efficient and Selective In Vivo Expression of mRNA in Spleen Tissue.
    Ly Porosk, Heleri Heike Härk, Piret Arukuusk, Uku Haljasorg, Pärt Peterson, Kaido Kurrikoff.
      mRNA-based therapeutics are presently one of the nucleic acid-based therapeutics with a high potential for extraordinary success as preventive vaccines. Current applications with mRNA therapeutics rely on lipid nanoparticle (LNP) mediated delivery of nucleic acids. In order to achieve the transition from preventive to therapeutic vaccines, there is a challenge of delivering the mRNA into non-hepatic tissues, especially into lymphoid tissues such as the spleen and lymph nodes. In this work, we characterize new cell-penetrating peptides NF424 and NF436 that exhibit preferential delivery of mRNA into the spleen after a single i.v. injection, without the use of any active targeting mechanisms. We show that between the spleen, liver, and the lungs, >95% of mRNA expression arises in the spleen tissue and the majority of expression occurs in the dendritic cells. The cell-penetrating peptides NF424 and NF436 represent promising candidates for cancer immunotherapeutic applications with tumor antigens.
    Keywords:  cell-penetrating peptides; freeze-dried nanoparticles; gene delivery; in vivo transfection; mRNA; mRNA delivery; pDNA; tissue specific expression
    DOI:  https://doi.org/10.3390/pharmaceutics15030952
  4. Polymers (Basel). 2023 Mar 18. pii: 1518. [Epub ahead of print]15(6):
    A New Optimization Strategy of Highly Branched Poly(β-Amino Ester) for Enhanced Gene Delivery: Removal of Small Molecular Weight Components.
    Yinghao Li, Xianqing Wang, Zhonglei He, Zishan Li, Melissa Johnson, Bei Qiu, Rijian Song, Sigen A, Irene Lara-Sáez, Jing Lyu, Wenxin Wang.
      Highly branched poly(β-amino ester) (HPAE) has become one of the most promising non-viral gene delivery vector candidates. When compared to other gene delivery vectors, HPAE has a broad molecular weight distribution (MWD). Despite significant efforts to optimize HPAE targeting enhanced gene delivery, the effect of different molecular weight (MW) components on transfection has rarely been studied. In this work, a new structural optimization strategy was proposed targeting enhanced HPAE gene transfection. A series of HPAE with different MW components was obtained through a stepwise precipitation approach and applied to plasmid DNA delivery. It was demonstrated that the removal of small MW components from the original HPAE structure could significantly enhance its transfection performance (e.g., GFP expression increased 7 folds at w/w of 10/1). The universality of this strategy was proven by extending it to varying HPAE systems with different MWs and different branching degrees, where the transfection performance exhibited an even magnitude enhancement after removing small MW portions. This work opened a new avenue for developing high-efficiency HPAE gene delivery vectors and provided new insights into the understanding of the HPAE structure-property relationship, which would facilitate the translation of HPAEs in gene therapy clinical applications.
    Keywords:  gene transfection; highly branched poly(β-amino ester); non-viral vector; pDNA delivery; polymer component; step-wise precipitation
    DOI:  https://doi.org/10.3390/polym15061518
  5. Bioconjug Chem. 2023 Mar 30.
    Lysine-Derived Charge-Altering Releasable Transporters: Targeted Delivery of mRNA and siRNA to the Lungs.
    Timothy R Blake, Ole A W Haabeth, Adrienne Sallets, Rebecca L McClellan, Trevor J Del Castillo, Jose G Vilches-Moure, Wilson C Ho, Paul A Wender, Ronald Levy, Robert M Waymouth.
      Targeted delivery of nucleic acid therapeutics to the lungs could transform treatment options for pulmonary disease. We have previously developed oligomeric charge-altering releasable transporters (CARTs) for in vivo mRNA transfection and demonstrated their efficacy for use in mRNA-based cancer vaccination and local immunomodulatory therapies against murine tumors. While our previously reported glycine-based CART-mRNA complexes (G-CARTs/mRNA) show selective protein expression in the spleen (mouse, >99%), here, we report a new lysine-derived CART-mRNA complex (K-CART/mRNA) that, without additives or targeting ligands, shows selective protein expression in the lungs (mouse, >90%) following systemic IV administration. We further show that by delivering siRNA using the K-CART, we can significantly decrease expression of a lung-localized reporter protein. Blood chemistry and organ pathology studies demonstrate that K-CARTs are safe and well-tolerated. We report on the new step economical, organocatalytic synthesis (two steps) of functionalized polyesters and oligo-carbonate-co-α-aminoester K-CARTs from simple amino acid and lipid-based monomers. The ability to direct protein expression selectively in the spleen or lungs by simple, modular changes to the CART structure opens fundamentally new opportunities in research and gene therapy.
    DOI:  https://doi.org/10.1021/acs.bioconjchem.3c00019
  6. Adv Mater. 2023 Mar 31. e2211105
    Molecular Chameleon Carriers for Nucleic Acid Delivery: The Sweet Spot Between Lipoplexes and Polyplexes.
    Sophie Thalmayr, Melina Grau, Lun Peng, Jana Pöhmerer, Ulrich Wilk, Paul Folda, Mina Yazdi, Eric Weidinger, Tobias Burghardt, Miriam Höhn, Ernst Wagner, Simone Berger.
      Taking advantage of effective intracellular delivery mechanisms of both cationizable lipids and polymers, highly potent double pH-responsive nucleic acid carriers were generated by combining at least two lipo amino fatty acids (LAFs) as hydrophobic cationizable motifs with hydrophilic cationizable aminoethylene units into novel sequence-defined molecules. The pH-dependent tunable polarity of the LAF was successfully implemented by inserting a central tertiary amine, which disrupts the hydrophobic character once protonated, resulting in pH-dependent structural and physical changes. This "molecular chameleon character" turned out to be advantageous for dynamic nucleic acid delivery via lipopolyplexes. By screening of different topologies (blocks, bundles, T-shapes, U-shapes), LAF types, and LAF/aminoethylene ratios, highly potent pDNA, mRNA, and siRNA carriers were identified, which were up to several hundred-fold more efficient than previous carrier generations and characterized by very fast transfection kinetics. mRNA lipopolyplexes maintained high transfection activity in cell culture even in the presence of ≥90% serum at an ultra-low mRNA dose of 3 picogram (∼2 nanoparticles/cell), and thus are comparable in potency to viral nanoparticles. Importantly, they showed great in vivo performance with high expression levels especially in spleen, tumor, lungs, and liver upon intravenous administration of 1-3 μg luciferase-encoding mRNA in mice. This article is protected by copyright. All rights reserved.
    Keywords:  endosomal escape; lipopolyplexes; mRNA; pDNA; pH-responsive; siRNA
    DOI:  https://doi.org/10.1002/adma.202211105
  7. Pharmaceutics. 2023 Mar 16. pii: 961. [Epub ahead of print]15(3):
    A New Strategy for Nucleic Acid Delivery and Protein Expression Using Biocompatible Nanohydrogels of Predefined Sizes.
    Lakshmanan Eswaran, Gila Kazimirsky, Ronen Yehuda, Gerardo Byk.
      We have developed new formulations of nanohydrogels (NHGs) complexed with DNA devoid of cell toxicity, which, together with their tuned sizes, makes them of great interest for delivering DNA/RNA for foreign protein expression. Transfection results demonstrate that, unlike classical lipo/polyplexes, the new NHGs can be incubated indefinitely with cells without apparent cellular toxicity, resulting in the high expression of foreign proteins for long periods of time. Although protein expression starts with a delay as compared to classical systems, it is sustained for a long period of time, even after passing cells without observation of toxicity. A fluorescently labelled NHG used for gene delivery was detected inside cells very early after incubation, but the protein expression was delayed by many days, demonstrating that there is a time-dependent release of genes from the NHGs. We suggest that this delay is due to the slow but continuous release of DNA from the particles concomitantly with slow but continuous protein expression. Additionally, results obtained after the in vivo administration of m-Cherry/NHG complexes indicated a delayed but prolonged expression of the marker gene in the tissue of administration. Overall, we have demonstrated gene delivery and foreign protein expression using GFP and m-Cherry marker genes complexed with biocompatible nanohydrogels.
    Keywords:  cationic nanohydrogels; non-viral gene delivery; polymerization; self-assembly
    DOI:  https://doi.org/10.3390/pharmaceutics15030961
  8. Acta Pharm Sin B. 2023 Mar;13(3): 955-966
    Nanoparticles (NPs)-mediated systemic mRNA delivery to reverse trastuzumab resistance for effective breast cancer therapy.
    Zhihui Dong, Zhuoshan Huang, Senlin Li, Ying Wang, Yandan Yao, Xianzhu Yang, Xiaoding Xu.
      Monoclonal antibody-based therapy has achieved great success and is now one of the most crucial therapeutic modalities for cancer therapy. The first monoclonal antibody authorized for treating human epidermal growth receptor 2 (HER2)-positive breast cancer is trastuzumab. However, resistance to trastuzumab therapy is frequently encountered and thus significantly restricts the therapeutic outcomes. To address this issue, tumor microenvironment (TME) pH-responsive nanoparticles (NPs) were herein developed for systemic mRNA delivery to reverse the trastuzumab resistance of breast cancer (BCa). This nanoplatform is comprised of a methoxyl-poly (ethylene glycol)-b-poly (lactic-co-glycolic acid) copolymer with a TME pH-liable linker (Meo-PEG-Dlink m -PLGA) and an amphiphilic cationic lipid that can complex PTEN mRNA via electrostatic interaction. When the long-circulating mRNA-loaded NPs build up in the tumor after being delivered intravenously, they could be efficiently internalized by tumor cells due to the TME pH-triggered PEG detachment from the NP surface. With the intracellular mRNA release to up-regulate PTEN expression, the constantly activated PI3K/Akt signaling pathway could be blocked in the trastuzumab-resistant BCa cells, thereby resulting in the reversal of trastuzumab resistance and effectively suppress the development of BCa.
    Keywords:  Cancer therapy; Monoclonal antibody therapy; Nanoparticle; Trastuzumab resistance; mRNA delivery
    DOI:  https://doi.org/10.1016/j.apsb.2022.09.021
  9. Acta Pharm Sin B. 2023 Mar;13(3): 942-954
    Sodium alginate coating simultaneously increases the biosafety and immunotherapeutic activity of the cationic mRNA nanovaccine.
    Xing Duan, Yi Zhang, Mengran Guo, Na Fan, Kepan Chen, Shugang Qin, Wen Xiao, Qian Zheng, Hai Huang, Xiawei Wei, Yuquan Wei, Xiangrong Song.
      The extraordinary advantages associated with mRNA vaccines, including their high efficiency, relatively low severity of side effects, and ease of manufacture, have enabled them to be a promising immunotherapy approach against various infectious diseases and cancers. Nevertheless, most mRNA delivery carriers have many disadvantages, such as high toxicity, poor biocompatibility, and low efficiency in vivo, which have hindered the widespread use of mRNA vaccines. To further characterize and solve these problems and develop a new type of safe and efficient mRNA delivery carrier, a negatively charged SA@DOTAP-mRNA nanovaccine was prepared in this study by coating DOTAP-mRNA with the natural anionic polymer sodium alginate (SA). Intriguingly, the transfection efficiency of SA@DOTAP-mRNA was significantly higher than that of DOTAP-mRNA, which was not due to the increase in cellular uptake but was associated with changes in the endocytosis pathway and the strong lysosome escape ability of SA@DOTAP-mRNA. In addition, we found that SA significantly increased the expression of LUC-mRNA in mice and achieved certain spleen targeting. Finally, we confirmed that SA@DOTAP-mRNA had a stronger antigen-presenting ability in E. G7-OVA tumor-bearing mice, dramatically inducing the proliferation of OVA-specific CLTs and ameliorating the antitumor effect. Therefore, we firmly believe that the coating strategy applied to cationic liposome/mRNA complexes is of potential research value in the field of mRNA delivery and has promising clinical application prospects.
    Keywords:  Cancer; Cationic liposome; DOTAP; Immunotherapy; Lysosomes escape; Negatively charged mRNA vaccine; Sodium alginate; Toxicity
    DOI:  https://doi.org/10.1016/j.apsb.2022.08.015
  10. Nat Biotechnol. 2023 Mar 30.
    Combinatorial design of nanoparticles for pulmonary mRNA delivery and genome editing.
    Bowen Li, Rajith Singh Manan, Shun-Qing Liang, Akiva Gordon, Allen Jiang, Andrew Varley, Guangping Gao, Robert Langer, Wen Xue, Daniel Anderson.
      The expanding applications of nonviral genomic medicines in the lung remain restricted by delivery challenges. Here, leveraging a high-throughput platform, we synthesize and screen a combinatorial library of biodegradable ionizable lipids to build inhalable delivery vehicles for messenger RNA and CRISPR-Cas9 gene editors. Lead lipid nanoparticles are amenable for repeated intratracheal dosing and could achieve efficient gene editing in lung epithelium, providing avenues for gene therapy of congenital lung diseases.
    DOI:  https://doi.org/10.1038/s41587-023-01679-x
  11. Nano Lett. 2023 Mar 27.
    Mechanism-Driven Technology Development for Solving the Intracellular Delivery Problem of Hard-To-Transfect Cells.
    Wanchuan Ding, Xuan Yang, Huoyue Lin, Zixing Xu, Jun Wang, Jie Dai, Can Xu, Feng Chen, Xiaowei Wen, Weiran Chai, Gang Ruan.
      The so-called "hard-to-transfect cells" are well-known to present great challenges to intracellular delivery, but detailed understandings of the delivery behaviors are lacking. Recently, we discovered that vesicle trapping is a likely bottleneck of delivery into a type of hard-to-transfect cells, namely, bone-marrow-derived mesenchymal stem cells (BMSCs). Driven by this insight, herein, we screened various vesicle trapping-reducing methods on BMSCs. Most of these methods failed in BMSCs, although they worked well in HeLa cells. In stark contrast, coating nanoparticles with a specific form of poly(disulfide) (called PDS1) nearly completely circumvented vesicle trapping in BMSCs, by direct cell membrane penetration mediated by thiol-disulfide exchange. Further, in BMSCs, PDS1-coated nanoparticles dramatically enhanced the transfection efficiency of plasmids of fluorescent proteins and substantially improved osteoblastic differentiation. In addition, mechanistic studies suggested that higher cholesterol content in plasma membranes of BMSCs might be a molecular-level reason for the greater difficulty of vesicle escape in BMSCs.
    Keywords:  gene delivery; nanomedicine; quantum dot; regenerative medicine; stem cell; tissue engineering; transfection
    DOI:  https://doi.org/10.1021/acs.nanolett.2c04834
  12. Pharmaceutics. 2023 Mar 17. pii: 974. [Epub ahead of print]15(3):
    Plant-Derived Extracellular Vesicles as a Delivery Platform for RNA-Based Vaccine: Feasibility Study of an Oral and Intranasal SARS-CoV-2 Vaccine.
    Margherita A C Pomatto, Chiara Gai, Federica Negro, Lucia Massari, Maria Chiara Deregibus, Cristina Grange, Francesco Giuseppe De Rosa, Giovanni Camussi.
      Plant-derived extracellular vesicles (EVs) may represent a platform for the delivery of RNA-based vaccines, exploiting their natural membrane envelope to protect and deliver nucleic acids. Here, EVs extracted from orange (Citrus sinensis) juice (oEVs) were investigated as carriers for oral and intranasal SARS-CoV-2 mRNA vaccine. oEVs were efficiently loaded with different mRNA molecules (coding N, subunit 1 and full S proteins) and the mRNA was protected from degrading stress (including RNase and simulated gastric fluid), delivered to target cells and translated into protein. APC cells stimulated with oEVs loaded with mRNAs induced T lymphocyte activation in vitro. The immunization of mice with oEVs loaded with S1 mRNA via different routes of administration including intramuscular, oral and intranasal stimulated a humoral immune response with production of specific IgM and IgG blocking antibodies and a T cell immune response, as suggested by IFN-γ production by spleen lymphocytes stimulated with S peptide. Oral and intranasal administration also triggered the production of specific IgA, the mucosal barrier in the adaptive immune response. In conclusion, plant-derived EVs represent a useful platform for mRNA-based vaccines administered not only parentally but also orally and intranasally.
    Keywords:  COVID-19; RNA; SARS; delivery; exosome; extracellular vesicle; intranasal; oral; plant; vaccine
    DOI:  https://doi.org/10.3390/pharmaceutics15030974
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