bims-novged 2023-03-19 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023–03–19
nine papers selected by
the Merkel lab, Ludwig-Maximilians University

Targeted siRNA lipid nanoparticles for the treatment of KRAS-mutant tumors.
The Effect of Cryoprotectants and Storage Conditions on the Transfection Efficiency, Stability and Safety of Lipid-Based Nanoparticles for mRNA and DNA Delivery.
Precise and systematic end group chemistry modifications on PAMAM and poly(l-lysine) dendrimers to improve cytosolic delivery of mRNA.
MC3/SAINT-O-Somes, a novel liposomal delivery system for efficient and safe delivery of siRNA into endothelial cells.
Biomimetic gene editing system for precise tumor cell reprogramming and augmented tumor therapy.
siRNA delivery against myocardial ischemia reperfusion injury mediated by reversibly camouflaged biomimetic nanocomplexes.
Pathogen Recognition-Driven Dendritic Cell-Specific Gene Silencing and Editing.
Unravelling the role of individual components in pBAE/polynucleotide polyplexes in the synthesis of tailored carriers for specific applications: on the road to rational formulations.
Protein adsorption determines pulmonary cell uptake of lipid-based nanoparticles.

J Control Release. 2023 Mar 13. pii: S0168-3659(23)00183-9. [Epub ahead of print]

Targeted siRNA lipid nanoparticles for the treatment of KRAS-mutant tumors.

Shubaash Anthiya, Süleyman Can Öztürk, Hamdullah Yanik, Ece Tavukcuoglu, Adem Sahin, Dhrubajyoti Datta, Klaus Charisse, David Moreira Álvarez, Mabel Loza, Alfonso Calvo, Einar Sulheim, Simon Loevenich, Geir Klinkenberg, Ruth Schmid, Muthiah Manoharan, Güneş Esendağlı, Maria Jose Alonso.

  K-RAS is a highly relevant oncogene that is mutated in approximately 90% of pancreatic cancers and 20-25% of lung adenocarcinomas. The aim of this work was to develop a new anti-KRAS siRNA therapeutic strategy through the engineering of functionalized lipid nanoparticles (LNPs). To do this, first, a potent pan anti-KRAS siRNA sequence was chosen from the literature and different chemical modifications of siRNA were tested for their transfection efficacy (KRAS knockdown) and anti-proliferative effects on various cancer cell lines. Second, a selected siRNA candidate was loaded into tLyp-1 targeted and non-targeted lipid nanoparticles (LNPs). The biodistribution and antitumoral efficacy of selected siRNA-loaded LNP-prototypes were evaluated in vivo using a pancreatic cancer murine model (subcutaneous xenograft CFPAC-1 tumors). Our results show that tLyp-1-tagged targeted LNPs have an enhanced accumulation in the tumor compared to non-targeted LNPs. Moreover, a significant reduction in the pancreatic tumor growth was observed when the anti-KRAS siRNA treatment was combined with a classical chemotherapeutic agent, gemcitabine. In conclusion, our work demonstrates the benefits of using a targeting approach to improve tumor accumulation of siRNA-LNPs and its positive impact on tumor reduction.

Keywords:  Combination therapy; KRAS; LNPs; Pancreatic cancer; RNA therapeutics; Targeted delivery; siRNA

DOI:  https://doi.org/10.1016/j.jconrel.2023.03.016
Adv Healthc Mater. 2023 Mar 12. e2203022

The Effect of Cryoprotectants and Storage Conditions on the Transfection Efficiency, Stability and Safety of Lipid-Based Nanoparticles for mRNA and DNA Delivery.

Konstantinos N Kafetzis, Natalia Papalamprou, Elisha McNulty, Kai X Thong, Yusuke Sato, Aleksandr Mironov, Atul Purohit, Philip J Welsby, Hideyoshi Harashima, Cynthia Yu-Wai-Man, Aristides D Tagalakis.

  Lipid-based nanoparticles have recently shown great promise, establishing themselves as the gold standard in delivering novel RNA therapeutics. However, research on the effects of storage on their efficacy, safety and stability is still lacking. Here, we explore the impact of storage temperature on two types of lipid-based nanocarriers, lipid nanoparticles (LNPs) and receptor-targeted nanoparticles (RTNs), loaded with either DNA or mRNA, and investigate the effects of different cryoprotectants on the stability and efficacy of the formulations. The medium-term stability of the nanoparticles was evaluated by monitoring their physicochemical characteristics, entrapment efficiency, and transfection efficiency, every 2 weeks over one month. We demonstrate that the use of cryoprotectants with nanocomplexes protects them against loss of function and degradation in all storage conditions. Moreover, we show that the addition of sucrose enables all nanoparticles to remain stable and maintain their efficacy for up to a month when stored at -80°C, regardless of cargo or type of nanoparticle. DNA-loaded nanoparticles also remained stable in a wider variety of storage conditions than mRNA-loaded ones. Importantly, these novel LNPs showed increased GFP expression that could signify their future use in gene therapies, beyond the established role of LNPs in RNA therapeutics. This article is protected by copyright. All rights reserved.

Keywords:  DNA; cryoprotectant; lipid nanoparticle; mRNA; stability

DOI:  https://doi.org/10.1002/adhm.202203022
J Control Release. 2023 Mar 12. pii: S0168-3659(23)00179-7. [Epub ahead of print]

Precise and systematic end group chemistry modifications on PAMAM and poly(l-lysine) dendrimers to improve cytosolic delivery of mRNA.

Fanny Joubert, Michael J Munson, Alan Sabirsh, Richard M England, Martin Hemmerling, Cameron Alexander, Marianne B Ashford.

  Here, we aimed to chemically modify PAMAM dendrimers using lysine as a site-selective anchor for successfully delivering mRNA while maintaining a low toxicity profile. PAMAM dendrimers were multi-functionalised by amidation reactions in a regioselective, quantitative and stepwise manner with carefully selected property-modifying surface groups. Alternatively, novel lysine-based dendrimers were prepared in the same manner with the aim to unlock their potential in gene delivery. The modified dendrimers were then formulated with Cy5-EGFP mRNA by bulk mixing via liquid handling robotics across different nitrogen to phosphate ratios. The resulting dendriplexes were characterised by size, charge, mRNA encapsulation, and mRNA binding affinity. Finally, their in-vitro delivery activity was systematically investigated across key cellular trafficking stages to relate chemical design to cellular effect. We demonstrate our findings in different cell lines and benchmarked relative to a commercially available transfection agent, jetPEI®. We demonstrate that specific surface modifications are required to generate small, reliable and well-encapsulated positively charged dendriplex complexes. Furthermore, we show that introduction of fusogenic groups is essential for driving endosomal escape and achieving cellular delivery and translation of mRNA in these cell lines.

Keywords:  Chemistry; Dendrimer; Formulation; Functional delivery; PAMAM; mRNA

DOI:  https://doi.org/10.1016/j.jconrel.2023.03.011
J Liposome Res. 2023 Mar 15. 1-10

MC3/SAINT-O-Somes, a novel liposomal delivery system for efficient and safe delivery of siRNA into endothelial cells.

Yutong He, Mees Barlag, Josée A Plantinga, Grietje Molema, Jan A A M Kamps.

  Increased understanding of chronic inflammatory diseases and the role of endothelial cell (EC) activation herein, have urged interest in sophisticated strategies to therapeutically intervene in activated EC to treat these diseases. Liposome-mediated delivery of therapeutic siRNA in inflammation-activated EC is such a strategy. In this study, we describe the design and characterisation of two liposomal siRNA delivery systems formulated with the cationic MC3 lipid or MC3/SAINT mixed lipids, referred to as MC3-O-Somes (MOS) and MC3/SAINT-O-Somes (MSS). The two formulations showed comparable physicochemical properties, except for better siRNA encapsulation efficiency in the MSS formulation. Antibody-mediated VCAM-1 targeting (AbVCAM-1) increased the association of the targeted MOS and MSS with activated EC, although the targeted MOS showed a significantly higher VCAM-1 specific association than the targeted MSS. AbVCAM-1 MSS containing RelA siRNA achieved significant downregulation of RelA expression, while AbVCAM-1 MOS containing RelA siRNA did not downregulate RelA expression in activated EC. Additionally, AbVCAM-1 MSS containing RelA siRNA showed low cytotoxicity in EC and at the same time prohibited endothelial inflammatory activation by reducing expression of cell adhesion molecules. The AbVCAM-1 MSS formulation is a novel siRNA delivery system based on a combination of the cationic lipids MC3 and SAINT, that shows good physicochemical characteristics, enhanced endothelial cell association, improved transfection activity, low toxicity and significant anti-inflammatory effect, thereby complying with the requirements for future in vivo investigations.

Keywords:  Cationic lipids; endothelial cell activation; endothelial cells; inflammation; liposomes; siRNA delivery

DOI:  https://doi.org/10.1080/08982104.2023.2187821
J Control Release. 2023 Mar 14. pii: S0168-3659(23)00187-6. [Epub ahead of print]

Biomimetic gene editing system for precise tumor cell reprogramming and augmented tumor therapy.

Lei Qiao, Min Gao, Xiaoqing Yi, Hui Peng, Ruijie Zhang, Wanqing Yao, Gengyun Sun, Xiaoyan He.

  The abnormal level of hypoxia-inducible factor-1 alpha (HIF-1α) is closely related to cancer metastasis and treatment resistance. CRISPR-Cas9-based gene editing technology has sparked profound hope to solve this issue by precise gene disruption, although the in vivo application remains hindered by the lack of a safe and efficient delivery strategy. Herein, we developed a cell membrane biomimetic core-shell system for light-controllable, precise gene editing. The inner core of the system comprises protamine for CRISPR-Cas9/sgRNA plasmid (pCas9) loading and calcium ions for efficient pCas9 transfection. The shell of the system is camouflaged by a cell membrane and modified with AS1411 aptamers for tumor targeting and photosensitizers to induce lysosomal escape and pCas9 release through reactive oxygen species production, thereby producing light-controllable enhanced gene editing. Neoplastic H1299 cells were reprogrammed using the biomimetic gene editing system upon laser irradiation with reduced VEGF and Vimentin expression, leading to enhanced antimetastatic effects. Genetic disruption of HIF-1α augmented the in vivo chemotherapeutic efficacy of paclitaxel. Our approach of using a membrane-camouflaged system combined with light augmentation provides a potential solution for the in vivo delivery of CRISPR-Cas9 as well as a feasible strategy for cancer therapy.

Keywords:  Biomimetic; Endo/lysosomal escape; Gene editing; Gene therapy; Targeting delivery

DOI:  https://doi.org/10.1016/j.jconrel.2023.03.020
Adv Mater. 2023 Mar 13. e2210691

siRNA delivery against myocardial ischemia reperfusion injury mediated by reversibly camouflaged biomimetic nanocomplexes.

Yang Zhou, Qiujun Liang, Xuejie Wu, Shanzhou Duan, Chenglong Ge, Huan Ye, Jianhui Lu, Rongying Zhu, Yongbing Chen, Fenghua Meng, Lichen Yin.

  siRNA-mediated management of myocardial ischemia reperfusion (IR) injury is greatly hampered by the inefficient myocardial enrichment and cardiomyocyte transfection. Herein, nanocomplexes (NCs) reversibly camouflaged with platelet-macrophage hybrid membrane (HM) were developed to efficiently deliver Sav1 siRNA (siSav1) into cardiomyocytes, suppressing the Hippo pathway and inducing cardiomyocyte regeneration. The biomimetic BSPC@HM NCs consisted of a cationic nano-core assembled from a membrane-penetrating helical polypeptide (P-Ben) and siSav1, a charge-reversal intermediate layer of poly(L -lysine)-cis-aconitic acid (PC), and an outer shell of HM. Due to HM-mediated inflammation homing and microthrombus targeting, intravenously injected BSPC@HM NCs could efficiently accumulate in the IR-injured myocardium, where the acidic inflammatory microenvironment triggered charge reversal of PC to shed off both HM and PC layers and allow the penetration of the exposed P-Ben/siSav1 NCs into cardiomyocytes. In rats and pigs, BSPC@HM NCs remarkably down-regulated Sav1 in IR-injured myocardium, promoted myocardium regeneration, suppressed myocardial apoptosis, and recovered cardiac functions. This study reports a bio-inspired strategy to overcome the multiple systemic barriers against myocardial siRNA delivery, and holds profound potentials for gene therapy against cardiac injuries. This article is protected by copyright. All rights reserved.

Keywords:  cardiomyocyte regeneration; charge reversal; hybrid cell membrane coating; myocardial ischemia reperfusion injury; siRNA delivery

DOI:  https://doi.org/10.1002/adma.202210691
Nano Lett. 2023 Mar 17.

Pathogen Recognition-Driven Dendritic Cell-Specific Gene Silencing and Editing.

Min Li, Han Zhou, Namei Wu, Wuxian Deng, Wang Dong, Xun Sun, Hang Liu, Zhigang Tian, Yucai Wang.

  Dendritic cells (DCs) play an essential role in both the induction of the immune response and the maintenance of immune tolerance, with any malfunction of DCs potentially causing several diseases. While gene-based therapy for DC manipulation is a promising approach, it remains challenging due to the lack of efficient delivery systems for DC targeting. Herein, we describe a novel bacterial nanomedicine (BNM) system for pathogen recognition-mediated DCs-specific gene silencing and gene editing. BNMs contain components from bacterial outer membranes and achieve efficient DC targeting through the recognition of pathogen-associated molecular patterns by pattern recognition receptors on DCs. The targeting efficiency of BNMs is reduced in DCs lacking toll-like receptor 4, which is responsible for recognizing lipopolysaccharide, a major component of the bacterial outer membrane. As a proof-of-concept demonstration, we present gene-based therapy mediated by BNMs for enhancing antigen cross-presentation in DCs, which generates a remarkable antitumor effect.

Keywords:  DC targeting; gene delivery; immunotherapy; nanoparticle; pathogen recognition

DOI:  https://doi.org/10.1021/acs.nanolett.3c00015
Nanoscale Adv. 2023 Mar 14. 5(6): 1611-1623

Unravelling the role of individual components in pBAE/polynucleotide polyplexes in the synthesis of tailored carriers for specific applications: on the road to rational formulations.

María Navalón-López, Aurora Dols-Perez, Santiago Grijalvo, Cristina Fornaguera, Salvador Borrós.

Oligopeptide end-modified poly(β-amino ester)s (OM-pBAEs) offer a means for the effective implementation of gene therapeutics in the near future. A fine-tuning of OM-pBAEs to meet application requirements is achieved by the proportional balance of oligopeptides used and provide gene carriers with high transfection efficacy, low toxicity, precise targeting, biocompatibility, and biodegradability. Understanding the influence and conformation of each building block at molecular and biological levels is therefore pivotal for further development and improvement of these gene carriers. Herein, we unmask the role of individual OM-pBAE components and their conformation in OM-pBAE/polynucleotide nanoparticles using a combination of fluorescence resonance energy transfer, enhanced darkfield spectral microscopy, atomic force microscopy, and microscale thermophoresis. We found that modifying the pBAE backbone with three end-terminal amino acids produces unique mechanical and physical properties for each combination. Higher adhesion properties are seen with arginine and lysine-based hybrid nanoparticles, while histidine provides an advantage in terms of construct stability. Our results shed light on the high potential of OM-pBAEs as gene delivery vehicles and provide insights into the influence of the nature of surface charges and the chemical nature of the pBAE modifications on their paths towards endocytosis, endosomal escape, and transfection.

DOI: https://doi.org/10.1039/d2na00800a
J Colloid Interface Sci. 2023 Mar 11. pii: S0021-9797(23)00407-1. [Epub ahead of print]641 36-47

Protein adsorption determines pulmonary cell uptake of lipid-based nanoparticles.

Santhni Subramaniam, Paul Joyce, Leigh Donnellan, Clifford Young, Anthony Wignall, Peter Hoffmann, Clive A Prestidge.

  The inhalable administration of lipid nanoparticles is an effective strategy for localised delivery of therapeutics against various lung diseases. Of this, improved intracellular delivery of pharmaceuticals for infectious disease and cancer management is of high significance. However, the influence of lipid nanoparticle composition and structure on uptake in pulmonary cell lines, especially in the presence of biologically relevant media is poorly understood. Here, the uptake of lamellar (liposomes) versus non-lamellar (cubosomes) lipid nanoparticles in macrophages and lung epithelial cells was quantified and the influence of bronchoalveolar lavage fluid (BALF), containing native pulmonary protein and surfactant molecules is determined. Cubosome uptake in both macrophages and epithelial cells was strongly mediated by a high percentage of molecular function regulatory and binding proteins present within the protein corona. In contrast, the protein corona did not influence the uptake of liposomes in epithelial cells. In macrophages, the proteins mediated a rapid internalisation, followed by exocytosis of liposomes after 6 h incubation. These findings on the influence of biological fluid in regulating lipid nanoparticle uptake mechanisms may guide future development of optimal intracellular delivery systems for therapeutics via the pulmonary route.

Keywords:  Cellular uptake; Lipid-based nanoparticle; Protein corona; Protein interaction; Pulmonary delivery

DOI:  https://doi.org/10.1016/j.jcis.2023.03.048