bims-novged 2021-12-19 papers

on Non-viral vectors for gene delivery

Issue of 2021–12–19
nine papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Optimization of phospholipid chemistry for improved lipid nanoparticle (LNP) delivery of messenger RNA (mRNA).
Mechanistic Studies of an Automated Lipid Nanoparticle Reveal Critical Pharmaceutical Properties Associated with Enhanced mRNA Functional Delivery In Vitro and In Vivo.
Cell-Penetrating Peptide-Modified Graphene Oxide Nanoparticles Loaded with Rictor siRNA for the Treatment of Triple-Negative Breast Cancer.
Non-viral siRNA transfection of primary mesenchymal stromal cells (MSCs): Assessment of tyrosine-modified PEI and PPI efficacy and biocompatibility.
The role of nanoparticle format and route of administration on self-amplifying mRNA vaccine potency.
Bridging mRNA and Polycation using RNA Oligonucleotide Derivatives Improves the Robustness of Polyplex Micelles for Efficient mRNA Delivery.
Efficient CRISPR-Cas9-based knockdown of RUNX2 to induce chondrogenic differentiation of stem cells.
Together is Better: mRNA Co-Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co-Delivery and Protein Expression on the Single Cell Level.
Biomimetic nanoparticles deliver mRNAs encoding costimulatory receptors and enhance T cell mediated cancer immunotherapy.

Biomater Sci. 2021 Dec 14.

Optimization of phospholipid chemistry for improved lipid nanoparticle (LNP) delivery of messenger RNA (mRNA).

Ester Álvarez-Benedicto, Lukas Farbiak, Martha Márquez Ramírez, Xu Wang, Lindsay T Johnson, Osamah Mian, Erick D Guerrero, Daniel J Siegwart.

Lipid nanoparticles (LNPs) have been established as an essential platform for nucleic acid delivery. Efforts have led to the development of vaccines that protect against SARS-CoV-2 infection using LNPs to deliver messenger RNA (mRNA) coding for the viral spike protein. Out of the four essential components that comprise LNPs, phospholipids represent an underappreciated opportunity for fundamental and translational study. We investigated this avenue by systematically modulating the identity of the phospholipid in LNPs with the goal of identifying specific moieties that directly enhance or hinder delivery efficacy. Results indicate that phospholipid chemistry can enhance mRNA delivery by increasing membrane fusion and enhancing endosomal escape. Phospholipids containing phosphoethanolamine (PE) head groups likely increase endosomal escape due to their fusogenic properties. Additionally, it was found that zwitterionic phospholipids mainly aided liver delivery, whereas negatively charged phospholipids changed the tropism of the LNPs from liver to spleen. These results demonstrate that the choice of phospholipid plays a role intracellularly by enhancing endosomal escape, while also driving organ tropism in vivo. These findings were then applied to Selective Organ Targeting (SORT) LNPs to manipulate and control spleen-specific delivery. Overall, selection of the phospholipid in LNPs provides an important handle to design and optimize LNPs for improved mRNA delivery and more effective therapeutics.

DOI: https://doi.org/10.1039/d1bm01454d
Small. 2021 Dec 16. e2105832

Mechanistic Studies of an Automated Lipid Nanoparticle Reveal Critical Pharmaceutical Properties Associated with Enhanced mRNA Functional Delivery In Vitro and In Vivo.

Lili Cui, Morag R Hunter, Silvia Sonzini, Sara Pereira, Steven M Romanelli, Kai Liu, Weimin Li, Lihuan Liang, Bin Yang, Najet Mahmoudi, Arpan S Desai.

  Recently, lipid nanoparticles (LNPs) have attracted attention due to their emergent use for COVID-19 mRNA vaccines. The success of LNPs can be attributed to ionizable lipids, which enable functional intracellular delivery. Previously, the authors established an automated high-throughput platform to screen ionizable lipids and identified that the LNPs generated using this automated technique show comparable or increased mRNA functional delivery in vitro as compared to LNPs prepared using traditional microfluidics techniques. In this study, the authors choose one benchmark lipid, DLin-MC3-DMA (MC3), and investigate whether the automated formulation technique can enhance mRNA functional delivery in vivo. Interestingly, a 4.5-fold improvement in mRNA functional delivery in vivo by automated LNPs as compared to LNPs formulated by conventional microfluidics techniques, is observed. Mechanistic studies reveal that particles with large size accommodate more mRNA per LNP, possess more hydrophobic surface, are more hemolytic, bind a larger protein corona, and tend to accumulate more in macropinocytosomes, which may quantitatively benefit mRNA cytosolic delivery. These data suggest that mRNA loading per particle is a critical factor that accounts for the enhanced mRNA functional delivery of automated LNPs. These mechanistic findings provide valuable insight underlying the enhanced mRNA functional delivery to accelerate future mRNA LNP product development.

Keywords:  hydrophobic surfaces; lipid nanoparticles; mRNA loading; macropinocytosis; particle size

DOI:  https://doi.org/10.1002/smll.202105832
Drug Des Devel Ther. 2021 ;15 4961-4972

Cell-Penetrating Peptide-Modified Graphene Oxide Nanoparticles Loaded with Rictor siRNA for the Treatment of Triple-Negative Breast Cancer.

Yun-Yun Yang, Wei Zhang, Hui Liu, Jun-Jie Jiang, Wen-Jie Wang, Zheng-Yan Jia.

   Introduction: Breast cancer is a malignant tumor that seriously threatens women's life and health.
Methods: In this study, we proposed to use graphene nanoparticles loaded with siRNA that can silence Rictor molecules essential for the mammalian target of rapamycin (mTOR) complex 2 (mTORC2) complex to enhance gene delivery to tumor cells through modification of cell-penetrating peptide (CPP) for the treatment of breast cancer.
Results: Remarkably, we successfully synthesized graphene oxide (GO)/polyethyleneimine (PEI)/polyethylene glycol (PEG)/CPP/small interfering RNA (siRNA) system, and the results were observed by atomic force microscopy (AFM) and ultraviolet visible (UV-Vis) absorption spectra. The optimum mass ratio of siRNA to GO-PEI-PEG-CPP was 1:0.5. We screened out Rictor siRNA-2 from 9 candidates, which presented the highest inhibition rate, and this siRNA was selected for the subsequent experiments. We validated that Rictor siRNA-2 significantly reduced the Rictor expression in triple negative breast cancer (TNBC) cells. Confocal fluorescence microscope and flow cytometry analysis showed that GO-PEI-PEG-CPP/siRNA was able to be effectively uptake by TNBC cells. GO-PEI-PEG-CPP/siRNA improved the effect of siRNA on the inhibition of TNBC cell viability and the induction of TNBC cell apoptosis. The expression of Rictor and the phosphorylation of Akt and p70s6k were inhibited by GO-PEI-PEG-CPP/siRNA. Tumorigenicity analysis in nude mice showed that GO-PEI-PEG-CPP/siRNA significantly repressed the tumor growth of TNBC cells in vivo. The levels of ki-67 were repressed by GO-PEI-PEG-CPP/siRNA, and the apoptosis was induced by GO-PEI-PEG-CPP/siRNA in the system.
Discussion: Therefore, we concluded that CPP-modified GO nanoparticles loaded with Rictor siRNA significantly repressed TNBC progression by the inhibition of PI3K/Akt/mTOR signaling. Our finding provides a promising therapeutic strategy for the treatment of TNBC.

Keywords:  PI3K/Akt/mTOR signaling; Rictor; cell-penetrating peptide; graphene oxide; triple-negative breast cancer

DOI:  https://doi.org/10.2147/DDDT.S330059
Int J Pharm. 2021 Dec 08. pii: S0378-5173(21)01165-0. [Epub ahead of print]612 121359

Non-viral siRNA transfection of primary mesenchymal stromal cells (MSCs): Assessment of tyrosine-modified PEI and PPI efficacy and biocompatibility.

Sandra Noske, Michael Karimov, Max Hansen, Nathalie Zatula, Alexander Ewe, Achim Aigner.

  Mesenchymal stromal cells (MSCs) are multipotent cells derived from different sources and able to differentiate into distinct cell lineages. For their possible biomedical application, the "tuning" of MSCs also involves the specific knockdown of defined target genes. A major limitation, however, is the notoriously low transfection efficacy especially of primary MSCs. In this paper, we systemically analyze a large set of tyrosine-modified linear or branched low molecular weight polyethylenimines (PEIs) of different sizes, as well as the tyrosine-modified polypropylenimine dendrimer PPI-G4, for their capacity of non-viral siRNA transfection into umbilical cord-derived MSCs from two different donors. Knockdown efficacies are determined on the molecular level and confirmed in functional assays. Beyond the determination of cell viabilities, acute cytotoxicity, induction of apoptosis/necrosis and mitochondrial membrane alterations are also studied. On the molecular level, caspase activation, ROS induction and genotoxic effects are analyzed. Major differences are observed between the various tyrosine-modified PEIs, with some candidates showing high knockdown efficacy and biocompatibility. PPI-G4-Y dendrimers, however, are identified as most efficient for siRNA transfection into MSCs. PPI-G4-Y/siRNA nanoparticles lead to particularly high gene knockdown, without cytotoxic and genotoxic effects on the cellular and molecular level, and are thus particularly well-suited for the tuning of MSCs.

Keywords:  PEI; PPI; Primary stromal cells; Tyrosine-modified PEI/PPI; siRNA transfection

DOI:  https://doi.org/10.1016/j.ijpharm.2021.121359
J Control Release. 2021 Dec 09. pii: S0168-3659(21)00659-3. [Epub ahead of print]

The role of nanoparticle format and route of administration on self-amplifying mRNA vaccine potency.

Giulia Anderluzzi, Gustavo Lou, Stuart Woods, Signe Tandrup Schmidt, Simona Gallorini, Michela Brazzoli, Russell Johnson, Craig W Roberts, Derek T O'Hagan, Barbara C Baudner, Yvonne Perrie.

  The efficacy of RNA-based vaccines has been recently demonstrated, leading to the use of mRNA-based COVID-19 vaccines. The application of self-amplifying mRNA within these formulations may offer further enhancement to these vaccines, as self-amplifying mRNA replicons enable longer expression kinetics and more potent immune responses compared to non-amplifying mRNAs. To investigate the impact of administration route on RNA-vaccine potency, we investigated the immunogenicity of a self-amplifying mRNA encoding the rabies virus glycoprotein encapsulated in different nanoparticle platforms (solid lipid nanoparticles (SLNs), polymeric nanoparticles (PNPs) and lipid nanoparticles (LNPs)). These were administered via three different routes: intramuscular, intradermal and intranasal. Our studies in a mouse model show that the immunogenicity of our 4 different saRNA vaccine formulations after intramuscular or intradermal administration was initially comparable; however, ionizable LNPs gave higher long-term IgG responses. The clearance of all 4 of the nanoparticle formulations from the intramuscular or intradermal administration site was similar. In contrast, immune responses generated after intranasal was low and coupled with rapid clearance for the administration site, irrespective of the formulation. These results demonstrate that both the administration route and delivery system format dictate self-amplifying RNA vaccine efficacy.

Keywords:  Immunogenicity; Lipid nanoparticles; Polymeric nanoparticles; RNA vaccines; Route of administration; Self-amplifying RNA; Solid lipid nanoparticles; saRNA

DOI:  https://doi.org/10.1016/j.jconrel.2021.12.008
Adv Healthc Mater. 2021 Dec 16. e2102016

Bridging mRNA and Polycation using RNA Oligonucleotide Derivatives Improves the Robustness of Polyplex Micelles for Efficient mRNA Delivery.

Naoto Yoshinaga, Satoshi Uchida, Anjaneyulu Dirisala, Mitsuru Naito, Kyoko Koji, Kensuke Osada, Horacio Cabral, Kazunori Kataoka.

  Polyplex for mRNA delivery requires strong yet reversible association between mRNA and polycation for extracellular robustness and selective intracellular disintegration. Herein, RNA oligonucleotide (OligoRNA) derivatives that bridge mRNA and polycation are developed to stabilize polyplex micelles (PMs). A set of the OligoRNAs introduced with a polyol moiety in their 5' end is designed to hybridize to fixed positions along mRNA strand. After PM preparation from the hybridized mRNA and poly(ethylene glycol) (PEG)-polycation block copolymer derivatized with phenylboronic acid (PBA) moieties in its cationic segment, PBA moieties form reversible phenylboronate ester linkages with a polyol moiety at 5' end of OligoRNAs and a diol moiety at their 3' end ribose, in the PM core. The OligoRNAs work as a node to bridge ionically complexed mRNA and polycation, thereby improving PM stability against polyion exchange reaction and ribonuclease attack in extracellular environment. After cellular uptake, intracellular high concentration of adenosine triphosphate (ATP) triggers the cleavage of phenylboronate ester linkages, resulting in mRNA release from PM. Ultimately, the PM provides efficient mRNA introduction in cultured cells and mouse lungs after intratracheal administration, demonstrating the potential of the bridging strategy in polyplex-based mRNA delivery. This article is protected by copyright. All rights reserved.

Keywords:  ATP-responsiveness; Block copolymer; Phenylboronic acid; Polyplex micelle; mRNA delivery; mRNA engineering

DOI:  https://doi.org/10.1002/adhm.202102016
Biomater Sci. 2021 Dec 14.

Efficient CRISPR-Cas9-based knockdown of RUNX2 to induce chondrogenic differentiation of stem cells.

Hye Jin Kim, Jong Min Park, Sujin Lee, Hui Bang Cho, Ji-In Park, Jae-Hwan Kim, Ji Sun Park, Keun-Hong Park.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system recognizes and deletes specific nucleotide sequences in cells for gene editing. This study aimed to edit and knockdown the RUNX2 gene, a key transcription factor that is directly involved in all stages of stem cell differentiation into osteoblasts. The RUNX2 gene was depleted using the CRISPR-Cas9 system to inhibit osteoblast differentiation of stem cells. shRNA vectors targeting RUNX2 were used as a control. The surface of nanoparticles (NPs) was coated with the cationic polymer linear polyethyleneimine. Thereafter, negatively charged CRISPR-Cas9 and shRNA vectors were complexed with positively charged NPs via ionic interactions. Several analytical methods were used to determine the size, surface charge, and morphology of NPs and to characterize the complexed genes. NPs complexed with CRISPR-Cas9 and shRNA vectors were delivered into human mesenchymal stem cells (hMSCs) via endocytosis. The mRNA and protein expression patterns of various genes in hMSCs were measured over time following internalization of NPs complexed with CRISPR-Cas9 and shRNA vectors in two- and three-dimensional culture systems. Knockdown of the RUNX2 gene decreased osteogenic differentiation and increased chondrogenic differentiation of hMSCs. As a result of investigating the efficiency of NPs complexed with CRISPR-Cas9 (CASP-NPs), Runx2 effectively knocked down in mesenchymal stem cells to enhance differentiation into chondrocytes, therefore CASP-NPs proved to be an effective gene carrier in hMSCs.

DOI: https://doi.org/10.1039/d1bm01716k
Adv Sci (Weinh). 2021 Dec 16. e2102072

Together is Better: mRNA Co-Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co-Delivery and Protein Expression on the Single Cell Level.

Heyang Zhang, Jeroen Bussmann, Florian H Huhnke, Joke Devoldere, An-Katrien Minnaert, Wim Jiskoot, Friedhelm Serwane, Joachim Spatz, Magnus Röding, Stefaan C De Smedt, Kevin Braeckmans, Katrien Remaut.

  Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co-deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle-lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre-defined ratio. A mixture of lipoplexes containing only one mRNA type (single-lipoplexes), however, seem to follow the "first come - first serve" principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single-cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle- and single-lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle-lipoplexes outperformed single-lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells.

Keywords:  cellular uptake; mingle/single-mRNA lipoplex; protein expression; single cell; theoretical modeling

DOI:  https://doi.org/10.1002/advs.202102072
Nat Commun. 2021 Dec 14. 12(1): 7264

Biomimetic nanoparticles deliver mRNAs encoding costimulatory receptors and enhance T cell mediated cancer immunotherapy.

Wenqing Li, Xinfu Zhang, Chengxiang Zhang, Jingyue Yan, Xucheng Hou, Shi Du, Chunxi Zeng, Weiyu Zhao, Binbin Deng, David W McComb, Yuebao Zhang, Diana D Kang, Junan Li, William E Carson, Yizhou Dong.

Antibodies targeting costimulatory receptors of T cells have been developed for the activation of T cell immunity in cancer immunotherapy. However, costimulatory molecule expression is often lacking in tumor-infiltrating immune cells, which can impede antibody-mediated immunotherapy. Here, we hypothesize that delivery of costimulatory receptor mRNA to tumor-infiltrating T cells will enhance the antitumor effects of antibodies. We first design a library of biomimetic nanoparticles and find that phospholipid nanoparticles (PL1) effectively deliver costimulatory receptor mRNA (CD137 or OX40) to T cells. Then, we demonstrate that the combination of PL1-OX40 mRNA and anti-OX40 antibody exhibits significantly improved antitumor activity compared to anti-OX40 antibody alone in multiple tumor models. This treatment regimen results in a 60% complete response rate in the A20 tumor model, with these mice being resistant to rechallenge by A20 tumor cells. Additionally, the combination of PL1-OX40 mRNA and anti-OX40 antibody significantly boosts the antitumor immune response to anti-PD-1 + anti-CTLA-4 antibodies in the B16F10 tumor model. This study supports the concept of delivering mRNA encoding costimulatory receptors in combination with the corresponding agonistic antibody as a strategy to enhance cancer immunotherapy.

DOI: https://doi.org/10.1038/s41467-021-27434-x