bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2023–04–09
thirteen papers selected by
the Merkel lab, Ludwig-Maximilians University
  1. On the mechanism of tissue-selective gene delivery by lipid nanoparticles.
  2. Intracellular trafficking kinetics of nucleic acid escape from lipid nanoparticles via fluorescence imaging.
  3. Recent progress in polymeric gene vectors: Delivery mechanisms, molecular designs, and applications.
  4. High-throughput screens identify a lipid nanoparticle that preferentially delivers mRNA to human tumors in vivo.
  5. Superior gene transfection efficiency in triple negative breast cancer by RAFT-mediated amino acid-based cationic diblock copolymers.
  6. Nanotechnology-enabled gene delivery for cancer and other genetic diseases.
  7. Updated landscape of inhaled medications to combat COVID-19.
  8. [Development of an Inhalation Dry Powder Preparation Method without Heat-drying Process].
  9. Self-driving laboratories: A paradigm shift in nanomedicine development.
  10. Untangling the Interactions between Anionic Polystyrene Nanoparticles and Lipid Membranes Using Laurdan Fluorescence Spectroscopy and Molecular Simulations.
  11. Ionizable lipids penetrate phospholipid bilayers with high phase transition temperatures: perspectives from free energy calculations.
  12. Knowledge-Based Design of Multifunctional Polymeric Nanoparticles.
  13. Non-replicating synthetic mRNA vaccines: A journey through the European (Journal of Immunology) history.
  1. J Control Release. 2023 Apr 05. pii: S0168-3659(23)00243-2. [Epub ahead of print]
    On the mechanism of tissue-selective gene delivery by lipid nanoparticles.
    Seigo Kimura, Hideyoshi Harashima.
      The era of nucleic acid nanomedicine has arrived, as evidenced by Patisiran, a small interfering RNA (siRNA) encapsulated lipid nanoparticle (LNP), and mRNA-loaded LNPs used in COVID-19 vaccines. The diversity of nano-designs for delivering nucleic acid molecules tested in Phase II/III clinical trials reflects the potential of these technologies. These breakthroughs in non-viral gene delivery, including the use of LNPs, have attracted substantial interest worldwide for developing more effective drugs. A next step in this field is to target tissues other than the liver, which requires significant research efforts and material development. However, mechanistic studies in this area are lacking. This study compares two types of LNPs with different tissue-selectivity for delivering plasmid DNA (pDNA), one being liver-selective and the other spleen-selective, in an effort to understand the mechanisms responsible for differences in gene expression of delivered genes. We observed little difference in the biodistribution of these two LNPs despite the 100-1000-fold differences in gene expression. We then quantified the amount of delivered pDNA and mRNA expression in each tissue by quantitative real-time PCR (qPCR) to evaluate various intracellular processes, such as nuclear delivery, transcription and translation. The results showed a >100-fold difference in the translation step but there were little differences in amount of pDNA delivered to the nucleus or the amount of mRNA expression for the two LNP deliveries. Our findings suggest that endogenous factors affect gene expression efficiency not the extent of biodistribution.
    Keywords:  Biodistribution; Cellular response; Gene delivery; Gene expression; Lipid nanoparticle; Plasmid DNA
    DOI:  https://doi.org/10.1016/j.jconrel.2023.03.052
  2. Curr Pharm Biotechnol. 2023 Apr 03.
    Intracellular trafficking kinetics of nucleic acid escape from lipid nanoparticles via fluorescence imaging.
    Christina M Bailey-Hytholt, Gregory Ulinski, Julia Dugas, Mohammed Haines, Mihael Lazebnik, Peter Piepenhagen, Isidro E Zarraga, Amey Bandekar.
       INTRODUCTION: Lipid nanoparticles (LNPs) are one of the most clinically advanced candidates for delivering nucleic acids to target cell populations, such as hepatocytes. Once LNPs are endocytosed, they must release their nucleic acid cargo into the cell cytoplasm. For delivering messenger RNA (mRNA), delivery into the cytosol is sufficient; however, for delivering DNA, there is an added diffusional barrier needed to facilitate nuclear uptake for transcription and therapeutic effect.
    METHOD: Here, we use fluorescence microscopy to investigate the intracellular fate of different LNP formulations to determine the kinetics of localization to endosomes and lysosomes. LNPs used in the studies were prepared via self-assembly using a NanoAssemblr for microfluidic mixing. As the content of polyethylene glycol (PEG) within the LNP formulation influences cellular uptake by hepatocyte cells, the content and hydrocarbon chain length within the formulation were assessed for their impact on intracellular trafficking. Standard LNPs were then formed using three commercially available ionizable lipids, Dlin-MC3-DMA (MC3), Dlin-KC2-DMA (KC2), and SS-OP. Plasmid DNA (pDNA) and mRNA were used, more specifically with a mixture of Cyanine 3 (Cy3)-labeled and green fluorescence protein (GFP) producing plasmid DNA (pDNA) as well as Cy5-labeled GFP producing mRNA. After formulation, LNPs were characterized for the encapsulation efficiency of the nucleic acid, hydrodynamic diameter, polydispersity, and zeta potential. All standard LNPs were ~100 nm in diameter and had neutral surface charge. All LNPs resulted in encapsulation efficiency greater than 70%. Confocal fluorescence microscopy was used for the intracellular trafficking studies, where LNPs were incubated with HuH-7 hepatocyte cells at times ranging from 0-48 h. The cells were antibody-stained for subcellular components, including nuclei, endosomes, and lysosomes.
    RESULT: Analysis was performed to quantify localization of pDNA to the endosomes and lysosomes. LNPs with 1.5 mol% PEG and a hydrocarbon chain C14 resulted in optimal endosomal escape and GFP production. Results from this study demonstrate that a higher percentage of C14 PEG leads to smaller LNPs with limited available phospholipid binding area for ApoE, resulting in decreased cellular uptake. We observed differences in the localization kinetics depending on the LNP formulation type for SS-OP, KC2, and MC3 ionizable lipids. The results also demonstrate the technique across different nucleic acid types, where mRNA resulted in more rapid and uniform GFP production compared to pDNA delivery.
    CONCLUSION: Here, we demonstrated the ability to track uptake and the sub-cellular fate of LNPs containing pDNA and mRNA, enabling improved screening prior to in vivo studies which would aid in formulation optimization.
    Keywords:  Fluorescence imaging; Intracellular trafficking; Lipid nanoparticles; Messenger RNA; Non-viral gene delivery; Nucleic acid; Plasmid DNA
    DOI:  https://doi.org/10.2174/1389201024666230403094238
  3. Biophys Rev (Melville). 2023 Mar;4(1): 011313
    Recent progress in polymeric gene vectors: Delivery mechanisms, molecular designs, and applications.
    Kemao Xiu, Jifeng Zhang, Jie Xu, Y Eugene Chen, Peter X Ma.
      Gene therapy and gene delivery have drawn extensive attention in recent years especially when the COVID-19 mRNA vaccines were developed to prevent severe symptoms caused by the corona virus. Delivering genes, such as DNA and RNA into cells, is the crucial step for successful gene therapy and remains a bottleneck. To address this issue, vehicles (vectors) that can load and deliver genes into cells are developed, including viral and non-viral vectors. Although viral gene vectors have considerable transfection efficiency and lipid-based gene vectors become popular since the application of COVID-19 vaccines, their potential issues including immunologic and biological safety concerns limited their applications. Alternatively, polymeric gene vectors are safer, cheaper, and more versatile compared to viral and lipid-based vectors. In recent years, various polymeric gene vectors with well-designed molecules were developed, achieving either high transfection efficiency or showing advantages in certain applications. In this review, we summarize the recent progress in polymeric gene vectors including the transfection mechanisms, molecular designs, and biomedical applications. Commercially available polymeric gene vectors/reagents are also introduced. Researchers in this field have never stopped seeking safe and efficient polymeric gene vectors via rational molecular designs and biomedical evaluations. The achievements in recent years have significantly accelerated the progress of polymeric gene vectors toward clinical applications.
    DOI:  https://doi.org/10.1063/5.0123664
  4. J Control Release. 2023 Apr 05. pii: S0168-3659(23)00255-9. [Epub ahead of print]
    High-throughput screens identify a lipid nanoparticle that preferentially delivers mRNA to human tumors in vivo.
    Sebastian G Huayamares, Melissa P Lokugamage, Regina Rab, Alejandro J Da Silva Sanchez, Hyejin Kim, Afsane Radmand, David Loughrey, Liming Lian, Yuning Hou, Bhagelu R Achyut, Annette Ehrhardt, Jeong S Hong, Cory D Sago, Kalina Paunovska, Elisa Schrader Echeverri, Daryll Vanover, Philip J Santangelo, Eric J Sorscher, E Dahlman.
      Lipid nanoparticles (LNPs) are a clinically relevant way to deliver therapeutic mRNA to hepatocytes in patients. However, LNP-mRNA delivery to end-stage solid tumors such as head and neck squamous cell carcinoma (HNSCC) remains more challenging. While scientists have used in vitro assays to evaluate potential nanoparticles for HNSCC delivery, high-throughput delivery assays performed directly in vivo have not been reported. Here we use a high-throughput LNP assay to evaluate how 94 chemically distinct nanoparticles delivered nucleic acids to HNSCC solid tumors in vivo. DNA barcodes were used to identify LNPHNSCC, a novel LNP for systemic delivery to HNSCC solid tumors. Importantly, LNPHNSCC retains tropism to HNSCC solid tumors while minimizing off-target delivery to the liver.
    Keywords:  DNA barcode; Lipid nanoparticles; cancer; mRNA delivery
    DOI:  https://doi.org/10.1016/j.jconrel.2023.04.005
  5. J Mater Chem B. 2023 Apr 04.
    Superior gene transfection efficiency in triple negative breast cancer by RAFT-mediated amino acid-based cationic diblock copolymers.
    Rima Saha, Satyajit Haldar, Subhashree Subhasmita Pradhan, Kuladip Jana, Kishor Sarkar.
      To date, the synthesis of efficient and safe gene carriers with low toxicity and appreciable gene transfection efficiency has been the major hurdle associated with non-viral gene carriers. Herein, we synthesized three amino acid-based diblock copolymers comprising glycine-leucine, leucine-phenyl alanine and glycine-phenyl alanine group containing blocks. The synthesis of all the diblock copolymers was confirmed by FTIR, 1H NMR, DLS and GPC techniques. All the polymers showed a high positive zeta potential value that varies from 45 ± 1 mV to 56 ± 1 mV, and the hydrodynamic size of the polymers varies from 250 ± 8 to 303 ± 14 nm. The three polymers showed negligible cytotoxicity compared with PEI (25 kDa) for MDA-MB-231 and NKE cells. Among all other polymers, P(HGN)n-b-P(HPN)m exhibited the highest biocompatibility with ∼70% cell viability at a concentration of 200 μg mL-1. Hemolysis data revealed that among all three polymers, P(HGN)n-b-P(HPN)m exhibited the highest blood compatibility, while up to a high concentration of 200 μg mL-1, it showed a very negligible amount (∼18%) of hemolysis. Most importantly, excellent gene complexation capability and good protection of pDNA against enzymatic degradation were observed with all three diblock copolymers. Interestingly, P(HGN)n-b-P(HPN)m/pDNA complex showed the smallest particle size (∼15 nm) and highest positive zeta potential as observed from TEM micrographs and DLS analysis, which probably results significantly higher level of cellular uptake and hence the highest transfection efficiency (∼85%) against MDA-MB-231 cells. Therefore, the diblock copolymer P(HGN)n-b-P(HPN)m with superior gene transfection efficiency in triple negative breast cancer may be an efficient non-viral vector for successful TNBC therapy in the future.
    DOI:  https://doi.org/10.1039/d2tb02681c
  6. Expert Opin Drug Deliv. 2023 Apr 05.
    Nanotechnology-enabled gene delivery for cancer and other genetic diseases.
    Tong Jiang, Karina Marie Gonzalez, Leyla Estrella Cordova, Jianqin Lu.
       INTRODUCTION: Despite gene therapy is ideal for genetic abnormality-related diseases, the easy degradation, poor targeting, and inefficiency in entering targeted cells are plaguing the effective delivery of gene therapy. Viral and non-viral vectors have been used for delivering gene therapeutics in vivo by safeguarding nucleic acid agents to target cells and to reach the specific intracellular location. A variety of nanotechnology-enabled safe and efficient systems have been successfully developed to improve the targeting ability for effective therapeutic delivery of genetic drugs.
    AREAS COVERED: In this review, we outline the multiple biological barriers associated with gene delivery process, and highlight recent advances to gene therapy strategy in vivo, including gene correction, gene silencing, gene activation and genome editing. We point out current developments and challenges exist of non-viral and viral vector systems in association with chemical and physical gene delivery technologies and their potential for the future.
    EXPERT OPINION: This review focuses on the opportunities and challenges to various gene therapy strategy, with specific emphasis on overcoming the challenges through the development of biocompatibility and smart gene vectors for potential clinical application.
    Keywords:  Gene therapy; drug delivery system; nanotechnology; non-viral vectors; viral vectors
    DOI:  https://doi.org/10.1080/17425247.2023.2200246
  7. Ther Deliv. 2023 Jan;14(1): 5-9
    Updated landscape of inhaled medications to combat COVID-19.
    Mural Quadros, Vivek Gupta.
      Plain language summary The COVID-19 pandemic has overwhelmed the healthcare systems worldwide. This calls for development of medicines that work effectively to decrease the death toll and severity of the infection. These medicines should be easy to administer to maintain patient compliance. Inhalation therapy is a needleless and painless route of administration that leads to fewer side effects. Various types of carriers are used for delivery of drugs via inhalation route. Vaccines can also be delivered via inhalation. Various researchers have studied vaccines via inhalation route, which have the potential to be translated into developing inhalable vaccines for COVID-19.
    Keywords:  COVID-19; inhalation therapy; lipid nanoparticles; nanocarriers; repurposing; vaccines
    DOI:  https://doi.org/10.4155/tde-2023-0003
  8. Yakugaku Zasshi. 2023 ;143(4): 353-358
    [Development of an Inhalation Dry Powder Preparation Method without Heat-drying Process].
    Takaaki Ito.
      Biopharmaceuticals, including therapeutic genes and proteins, are characterized by highly-targeted, specific action and flexible pharmacological design and have a rapidly growing market share; however, because of high molecular weight and low stability, injection is the most common delivery route of biopharmaceuticals. Thus, pharmaceutical innovations are required to provide alternative delivery routes for biopharmaceuticals. Pulmonary drug delivery via inhalation is a promising approach, particularly for targeting local diseases of the lung, because it can exert therapeutic effects in small doses and can noninvasively and directly deliver drugs to airway surfaces. However, biopharmaceutical inhalers must ensure that the biopharmaceuticals maintain their integrity as they are subjected to several types of physicochemical stress, such as hydrolysis, ultrasound, and heating, at various stages during the process from manufacturing to administration. In this symposium, I present a novel dry powder inhaler (DPI) preparation method without heat-drying, with the goal of developing biopharmaceutical DPIs. Spray-freeze-drying is a nonthermal drying technique that produces a powder with porous shapes; this powder has suitable inhalation characteristics for DPI. A model drug, plasmid DNA (pDNA), was stably prepared as a DPI using the spray-freeze-drying process. Under dry conditions, the powders maintained high inhalation characteristics and maintained pDNA integrity for 12 months. The powder induced pDNA expression in mouse lungs that exceeded at higher levels than the solution did. This novel preparation method is suitable for DPI preparation for various drugs and may help expand the clinical application of DPIs.
    Keywords:  dry powder inhalers; gene delivery; hyaluronic acid; spray-freeze-drying
    DOI:  https://doi.org/10.1248/yakushi.22-00170-3
  9. Matter. 2023 Apr 05. 6(4): 1071-1081
    Self-driving laboratories: A paradigm shift in nanomedicine development.
    Riley J Hickman, Pauric Bannigan, Zeqing Bao, Alán Aspuru-Guzik, Christine Allen.
      Nanomedicines have transformed promising therapeutic agents into clinically approved medicines with optimal safety and efficacy profiles. This is exemplified by the mRNA vaccines against COVID-19, which were made possible by lipid nanoparticle technology. Despite the success of nanomedicines to date, their design remains far from trivial, in part due to the complexity associated with their preclinical development. Herein, we propose a nanomedicine materials acceleration platform (NanoMAP) to streamline the preclinical development of these formulations. NanoMAP combines high-throughput experimentation with state-of-the-art advances in artificial intelligence (including active learning and few-shot learning) as well as a web-based application for data sharing. The deployment of NanoMAP requires interdisciplinary collaboration between leading figures in drug delivery and artificial intelligence to enable this data-driven design approach. The proposed approach will not only expedite the development of next-generation nanomedicines but also encourage participation of the pharmaceutical science community in a large data curation initiative.
    Keywords:  active learning; automation; drug delivery; high-throughput experimentation; lipid-based nanoparticles; liposomes; machine learning; materials acceleration platforms; nanomedicines; polymer nanoparticles; self-driving laboratories
    DOI:  https://doi.org/10.1016/j.matt.2023.02.007
  10. J Am Chem Soc. 2023 Apr 03.
    Untangling the Interactions between Anionic Polystyrene Nanoparticles and Lipid Membranes Using Laurdan Fluorescence Spectroscopy and Molecular Simulations.
    Laura A Kesner, Zeke A Piskulich, Qiang Cui, Zeev Rosenzweig.
      Several classes of synthetic nanoparticles (NPs) induce rearrangements of cell membranes that can affect membrane function. This paper describes the investigation of the interactions between polystyrene nanoparticles and liposomes, which serve as model cell membranes, using a combination of laurdan fluorescence spectroscopy and coarse-grained molecular dynamics (MD) simulations. The relative intensities of the gel-like and fluid fluorescent peaks of laurdan, which is embedded in the liposome membranes, are quantified from the areas of deconvoluted lognormal laurdan fluorescence peaks. This provides significant advantages in understanding polymer-membrane interactions. Our study reveals that anionic polystyrene NPs, which are not cross-linked, induce significant membrane rearrangement compared to other cationic or anionic NPs. Coarse-grained MD simulations demonstrate that polymer chains from the anionic polystyrene NP penetrate the liposome membrane. The inner leaflet remains intact throughout this process, though both leaflets show a decrease in lipid packing that is indicative of significant local rearrangement of the liposome membrane. These results are attributed to the formation of a hybrid gel made up of a combination of polystyrene (PS) and lipids that forces water molecules away from laurdan. Our study concludes that a combination of negative surface charge to interact electrostatically with positive charges on the membrane, a hydrophobic core to provide a thermodynamic preference for membrane association, and the ability to extend non-cross linked polymer chains into the liposome membrane are necessary for NPs to cause a significant rearrangement in the liposomes.
    DOI:  https://doi.org/10.1021/jacs.2c13403
  11. Chem Phys Lipids. 2023 Mar 30. pii: S0009-3084(23)00016-6. [Epub ahead of print] 105294
    Ionizable lipids penetrate phospholipid bilayers with high phase transition temperatures: perspectives from free energy calculations.
    Inna Ermilova, Jan Swenson.
      The efficacies of modern gene-therapies strongly depend on their contents. At the same time the most potent formulations might not contain the best compounds. In this work we investigated the effect of phospholipids and their saturation on the binding ability of (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA) to model membranes at the neutral pH. We discovered that DLin-MC3-DMA has affinity to the most saturated monocomponent lipid bilayer 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and an aversion to the unsaturated one 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The preference to a certain membrane was also well-correlated to the phase transition temperatures of phospholipid bilayers, and to their structural and dynamical properties. Additionally, in the case of the presence of DLin-MC3-DMA in the membrane with DOPC the ionizable lipid penetrated it, which indicates possible synergistic effects. Comparisons with other ionizable lipids were performed using a model lipid bilayer of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC). Particularly, the lipids heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102) and [(4-Hydroxybutyl) azanediyl] di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315) from modern mRNA-vaccines against COVID-19 were investigated and force fields parameters were derived for those new lipids. It was discovered that ALC-0315 binds strongest to the membrane, while DLin-MC3-DMA is not able to reside in the bilayer center. The ability to penetrate the membrane POPC by SM-102 and ALC-0315 can be related to their saturation, comparing to DLin-MC3-DMA.
    Keywords:  ALC-0315; DLin-MC3-DMA; Ionizable lipids; Lipid bilayer; Phase transition; SM-102
    DOI:  https://doi.org/10.1016/j.chemphyslip.2023.105294
  12. Handb Exp Pharmacol. 2023 Apr 06.
    Knowledge-Based Design of Multifunctional Polymeric Nanoparticles.
    Mira Behnke, Caroline T Holick, Antje Vollrath, Stephanie Schubert, Ulrich S Schubert.
      Conventional drug delivery systems (DDS) today still face several drawbacks and obstacles. High total doses of active pharmaceutical ingredients (API) are often difficult or impossible to deliver due to poor solubility of the API or undesired clearance from the body caused by strong interactions with plasma proteins. In addition, high doses lead to a high overall body burden, in particular if they cannot be delivered specifically to the target site. Therefore, modern DDS must not only be able to deliver a dose into the body, but should also overcome the hurdles mentioned above as examples. One of these promising devices are polymeric nanoparticles, which can encapsulate a wide range of APIs despite having different physicochemical properties. Most importantly, polymeric nanoparticles are tunable to obtain tailored systems for each application. This can already be achieved via the starting material, the polymer, by incorporating, e.g., functional groups. This enables the particle properties to be influenced not only specifically in terms of their interactions with APIs, but also in terms of their general properties such as size, degradability, and surface properties. In particular, the combination of size, shape, and surface modification allows polymeric nanoparticles to be used not only as a simple drug delivery device, but also to achieve targeting. This chapter discusses to what extent polymers can be designed to form defined nanoparticles and how their properties affect their performance.
    Keywords:  Active pharmaceutical ingredient; Drug delivery system; Formulation method; Nanoparticle; Polymer; Targeting
    DOI:  https://doi.org/10.1007/164_2023_649
  13. Eur J Immunol. 2023 Apr 07. e2249941
    Non-replicating synthetic mRNA vaccines: A journey through the European (Journal of Immunology) history.
    Steve Pascolo.
      The first worldwide article reporting that injections of synthetic non-replicating mRNA could be used as a vaccine, originated from a French team located in Paris was published in the European Journal of Immunology (EJI) in 1993. It relied on work conducted by several research groups in a handful of countries in the 1960s, who put forward the precise description of eukaryotic mRNA and the method to reproduce this molecule in vitro as well as how to transfect it into mammalian cells. Thereafter, the first industrial development of this technology began in 2000 in Germany, with the founding of CureVac, which stemmed from another description of a synthetic mRNA vaccine published in 2000 in EJI. The first clinical studies investigating mRNA vaccines in humans were performed as a collaboration between CureVac and the University of Tübingen in Germany as early as 2003. Finally, the first worldwide approved mRNA vaccine (an anti-COVID-19 vaccine) is based on the mRNA technologies developed by BioNTech since its foundation in 2008 in Mainz Germany, and earlier by the pioneering academic work of its founders. In addition to the past, present and future of mRNA-based vaccines, it is the aim of this article to present the geographical distribution of the early work, how the development of the technology was implemented by several independent and internationally distributed research teams, as well as the controversies on the optimal way to design/formulate and administer mRNA vaccines. This article is protected by copyright. All rights reserved.
    Keywords:  CleanCap; ivt mRNA; liposome; mRNA; vaccine
    DOI:  https://doi.org/10.1002/eji.202249941
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