bims-novged 2021-12-12 papers

on Non-viral vectors for gene delivery

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

Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells.
Uniform iron oxide nanoparticles reduce the required amount of polyethylenimine in the gene delivery to mesenchymal stem cells.
Chemically synthesized, self-assembling small interfering RNA-prohead RNA molecules trigger Dicer-independent gene silencing.
Enhanced nuclear gene delivery via integrating and streamlining intracellular pathway.
In Vitro Cellular Uptake and Transfection of Oligoarginine-Conjugated Glycol Chitosan/siRNA Nanoparticles.
Relationship between phosphorylamine-modification and molecular weight on transfection efficiency of chitosan.
Zwitterionic Phospholipidation of Cationic Polymers Facilitates Systemic mRNA Delivery to Spleen and Lymph Nodes.
Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale.
Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice.
pH-dependent behavior of ionizable cationic lipids in mRNA-carrying lipoplexes investigated by molecular dynamics simulations.
A phase 1 trial of lipid-encapsulated mRNA encoding a monoclonal antibody with neutralizing activity against Chikungunya virus.

AAPS J. 2021 12 06. 24(1): 8

Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells.

Purva Khare, Kandarp M Dave, Yashika S Kamte, Muthiah A Manoharan, Lauren A O'Donnell, Devika S Manickam.

  Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential delivery utility in either applications for the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that Cy5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX-the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile, whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered Cy5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells.

Keywords:  C12-200; lipidoid nanoparticle; neural cell; siRNA; transfection

DOI:  https://doi.org/10.1208/s12248-021-00653-2
Nanotechnology. 2021 Dec 06.

Uniform iron oxide nanoparticles reduce the required amount of polyethylenimine in the gene delivery to mesenchymal stem cells.

Donghang Xu, Yuanqin Su, Qianhao Xu, Ting Huang, Zhilan Chen, Tianyuan Zhang.

  Cationic polyethylenimine (PEI) is regarded as the "golden standard" of non-viral gene vectors. However, the superiority of PEI with high positive charge density also induces its major drawback of cytotoxicity, which restricts its application for an effective and safe gene delivery to stem cells. To redress this shortcoming, herein, a magnetic gene complex containing uniform iron oxide nanoparticles (UIONPs), plasmid DNA, and free PEI is prepared through electrostatic interactions for the gene delivery to bone marrow-derived mesenchymal stem cells (BM-MSCs). Results show that UIONPs dramatically promote the gene delivery to BM-MSCs using the assistance of magnetic force. In addition, decreasing the free PEI nitrogen to DNA phosphate (N/P) ratio from 10 to 6 has little adverse impact on the transgene expression levels (over 300 times than that of PEI alone at the N/P ratio of 6) and significantly reduces the cytotoxicity to BM-MSCs. Further investigations confirmed that the decrease of free PEI has little influence on the cellular uptake after applying external magnetic forces, but that the reduced positive charge density decreases the cytotoxicity. The present study demonstrates that the magnetic gene delivery not only contributes to the enhanced gene delivery efficiency but also helps to reduce required amount of PEI, providing a potential strategy for an efficient and safe gene delivery to stem cells.

Keywords:  cytotoxicity; gene delivery; iron oxide nanoparticles; mesenchymal stem cell; polyethylenimine

DOI:  https://doi.org/10.1088/1361-6528/ac4066
Chemistry. 2021 Dec 08.

Chemically synthesized, self-assembling small interfering RNA-prohead RNA molecules trigger Dicer-independent gene silencing.

Alyssa Hill, Daniël van Leeuwen, Verena Schlösser, Alok Behera, Bogdan Mateescu, Jonathan Hall.

  RNA interference (RNAi) mediated by small interfering RNA (siRNA) duplexes is a powerful therapeutic modality, but the translation of siRNAs from the bench into clinical application has been hampered by inefficient delivery in vivo . An innovative delivery strategy involves fusing siRNAs to a three-way junction (3WJ) motif derived from the phi29 bacteriophage prohead RNA (pRNA). Chimeric siRNA-3WJ molecules are presumed to enter the RNAi pathway via Dicer cleavage. Here, we fused siRNAs to the phi29 3WJ and two phylogenetically related 3WJs. We confirmed that the siRNA-3WJs are substrates for Dicer in vitro . However, our results reveal that siRNA-3WJs transfected into Dicer-deficient cell lines trigger potent gene silencing. Interestingly, siRNA-3WJs transfected into an Argonaute 2-deficient cell line also retain some gene silencing activity. siRNA-3WJs are most efficient when the antisense strand of the siRNA duplex is positioned 5' of the 3WJ (5'-siRNA-3WJ) relative to 3' of the 3WJ (3'-siRNA-3WJ). This work sheds light on the functional properties of siRNA-3WJs and offers a design rule for maximizing their potency in the human RNAi pathway.

Keywords:  RNA nanotechnology; packaging RNA; prohead RNA; small interfering RNA; three-way junction

DOI:  https://doi.org/10.1002/chem.202103995
J Control Release. 2021 Dec 02. pii: S0168-3659(21)00649-0. [Epub ahead of print]

Enhanced nuclear gene delivery via integrating and streamlining intracellular pathway.

Lian-Yu Qi, Yi Wang, Li-Fan Hu, Pu-Song Zhao, Hao-Yuan Yu, Lei Xing, Xiang-Dong Gao, Qing-Ri Cao, Hu-Lin Jiang.

  The essential challenge of gene therapy is to develop safe and efficient vectors that escort genes to target sites. However, due to the cumbersome workflow of gene transfection into cells, successive gene loss occurs. This leads to considerable reductions in nuclear gene uptake, eventually causing low gene expression. Herein, we designed a gene vector named CA3S2 (C: N,N'-cystamine-bis-acrylamide [CBA], A: agmatine dihydrochloride [Agm], S: 4-(2-aminoethyl) benzenesulfonamide [ABS]) with excellent gene transfection ability. This vector can promote gene delivery to the nucleus via enhanced endoplasmic reticulum (ER) targeting through integrating and streamlining of the complex intracellular pathway. Briefly, ABS endowed CA3S2/DNA nanoparticles with not only a natural ER-targeting tendency attributed to the caveolae-mediated pathway but also direct receptor-binding capacity on the ER surface. Agm enabled CA3S2 to enhance lysosomal escape and nuclear uptake ability. The gene delivery efficiency of CA3S2 was significantly better than that of polyethyleneimine 25K (PEI 25K). Therefore, CA3S2 is a promising gene carrier, and the ER-targeting strategy involving intracellular pathway integration and streamlining has potential for gene therapy.

Keywords:  Caveolae-mediated pathway; Endoplasmic reticulum-targeting; Gene therapy; Lysosomal escape; Nuclear delivery

DOI:  https://doi.org/10.1016/j.jconrel.2021.11.046
Polymers (Basel). 2021 Dec 01. pii: 4219. [Epub ahead of print]13(23):

In Vitro Cellular Uptake and Transfection of Oligoarginine-Conjugated Glycol Chitosan/siRNA Nanoparticles.

Eun-Ju Jeong, Jangwook Lee, Hyun-Seung Kim, Kuen-Yong Lee.

  Chitosan and its derivatives have been extensively utilized in gene delivery applications because of their low toxicity and positively charged characteristics. However, their low solubility under physiological conditions often limits their application. Glycol chitosan (GC) is a derivative of chitosan that exhibits excellent solubility in physiological buffer solutions. However, it lacks the positive characteristics of a gene carrier. Thus, we hypothesized that the introduction of oligoarginine peptide to GC could improve the formation of complexes with siRNA, resulting in enhanced uptake by cells and increased transfection efficiency in vitro. A peptide with nine arginine residues and 10 glycine units (R9G10) was successfully conjugated to GC, which was confirmed by infrared spectroscopy, 1H NMR spectroscopy, and elemental analysis. The physicochemical characteristics of R9G10-GC/siRNA complexes were also investigated. The size and surface charge of the R9G10-GC/siRNA nanoparticles depended on the amount of R9G10 coupled to the GC. In addition, the R9G10-GC/siRNA nanoparticles showed improved uptake in HeLa cells and enhanced in vitro transfection efficiency while maintaining low cytotoxicity determined by the MTT assay. Oligoarginine-modified glycol chitosan may be useful as a potential gene carrier in many therapeutic applications.

Keywords:  gene therapy; glycol chitosan; oligoarginine; siRNA

DOI:  https://doi.org/10.3390/polym13234219
Carbohydr Polym. 2022 Feb 01. pii: S0144-8617(21)01257-1. [Epub ahead of print]277 118870

Relationship between phosphorylamine-modification and molecular weight on transfection efficiency of chitosan.

Sibel Çelik, Mehmet Koray Gök, Kamber Demir, Serhat Pabuccuoğlu, Saadet Özgümüş.

  The aim of this study is both to design the chitosan (Chi) nanoparticles with different Mw containing the phosphoester bonds and increase their amino function for the transfection. The phosphorylamine-modification of Chi and depolymerized Chi (DChi) was realized using o-phosphorylethanolamine (o-PEA) and characterized, for the first time. The nanoparticles (nMChi and nMDChi) were prepared by ionic gelation and their particle size, polydispersity index (PDI), zeta potential, stability, gene binding capacity and cytotoxicity were examined. The effects of the Mw of Chi on the cytotoxicity, gene binding capacity, and in vitro transfection efficiency of the nanoparticles on Human Embryonic Kidney 293 (HEK293) cells were also examined. Green Fluorescent Protein Circular Plasmid DNA (pEGFN1) loaded nanoparticles (gnMChi and gnMDChi) were used in the transfection. This study showed that the Mw of phosphorylamine-modified Chi significantly affected the characteristics, cytotoxicity, gene binding capacity and transfection efficiency of the nanoparticles.

Keywords:  Chitosan; Gene carrier; Nanoparticle; Phosphorylation; Transfection

DOI:  https://doi.org/10.1016/j.carbpol.2021.118870
J Am Chem Soc. 2021 Dec 08.

Zwitterionic Phospholipidation of Cationic Polymers Facilitates Systemic mRNA Delivery to Spleen and Lymph Nodes.

Shuai Liu, Xu Wang, Xueliang Yu, Qiang Cheng, Lindsay T Johnson, Sumanta Chatterjee, Di Zhang, Sang M Lee, Yehui Sun, Ting-Chih Lin, John L Liu, Daniel J Siegwart.

Polymers represent a promising therapeutic platform for extrahepatic messenger RNA (mRNA) delivery but are hampered by low in vivo efficacy due to polyplex serum instability and inadequate endosomal escape following systemic administration. Here, we report the rational design and combinatorial synthesis of zwitterionic phospholipidated polymers (ZPPs) via cationic polymer postmodification by alkylated dioxaphospholane oxides to deliver mRNA to spleen and lymph nodes in vivo. This modular postmodification approach readily produces tunable zwitterionic species for serum resistance and introduces alkyl chains simultaneously to enhance endosomal escape, thereby transforming deficient cationic polymers to efficacious zwitterionic mRNA carriers without the need to elaborately synthesize functional monomers. ZPPs mediated up to 39 500-fold higher protein expression than their parent cationic counterparts in vitro and enabled efficacious mRNA delivery selectively in spleen and lymph nodes following intravenous administration in vivo. This zwitterionic phospholipidation methodology provides a versatile and generalizable postmodification strategy to introduce zwitterions into the side chains of cationic polymers, extending the utility of cationic polymer families for precise mRNA delivery and demonstrating substantial potential for immunotherapeutic applications.

DOI: https://doi.org/10.1021/jacs.1c09822
J Cell Biol. 2022 Feb 07. pii: e202110137. [Epub ahead of print]221(2):

Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale.

Prasath Paramasivam, Christian Franke, Martin Stöter, Andreas Höijer, Stefano Bartesaghi, Alan Sabirsh, Lennart Lindfors, Marianna Yanez Arteta, Anders Dahlén, Annette Bak, Shalini Andersson, Yannis Kalaidzidis, Marc Bickle, Marino Zerial.

Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.

DOI: https://doi.org/10.1083/jcb.202110137
Nat Commun. 2021 Dec 08. 12(1): 7101

Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice.

Eriya Kenjo, Hiroyuki Hozumi, Yukimasa Makita, Kumiko A Iwabuchi, Naoko Fujimoto, Satoru Matsumoto, Maya Kimura, Yuichiro Amano, Masataka Ifuku, Youichi Naoe, Naoto Inukai, Akitsu Hotta.

Genome editing therapy for Duchenne muscular dystrophy (DMD) holds great promise, however, one major obstacle is delivery of the CRISPR-Cas9/sgRNA system to skeletal muscle tissues. In general, AAV vectors are used for in vivo delivery, but AAV injections cannot be repeated because of neutralization antibodies. Here we report a chemically defined lipid nanoparticle (LNP) system which is able to deliver Cas9 mRNA and sgRNA into skeletal muscle by repeated intramuscular injections. Although the expressions of Cas9 protein and sgRNA were transient, our LNP system could induce stable genomic exon skipping and restore dystrophin protein in a DMD mouse model that harbors a humanized exon sequence. Furthermore, administration of our LNP via limb perfusion method enables to target multiple muscle groups. The repeated administration and low immunogenicity of our LNP system are promising features for a delivery vehicle of CRISPR-Cas9 to treat skeletal muscle disorders.

DOI: https://doi.org/10.1038/s41467-021-26714-w
Macromol Rapid Commun. 2021 Dec 07. e2100683

pH-dependent behavior of ionizable cationic lipids in mRNA-carrying lipoplexes investigated by molecular dynamics simulations.

Giovanni Settanni, Wolfgang Brill, Heinrich Haas, Friederike Schmid.

  Lipid-based nanoparticles and lipoplexes containing ionizable lipids in their formulation are among the most successful nanocarriers for mRNA-based therapies. At molecular level, the structure of these assemblies is still not fully understood, in particular regarding the role played by the ionizable lipid in the interactions with the RNA molecules. SAXS experiments have shown that lipoplexes based on the ionizable lipid 2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), under a certain range of conditions, have a lamellar structure, where lipid bilayers are separated by mRNA-rich layers, with an overall periodicity or spacing between 6.5 and 8.0 nm and a complex pH-dependence. Here, a multiscale molecular dynamics simulation approach is used to investigate the structure and dynamics of these lipoplex formulations at varying pH level, as well as, the effects of the introduction of mRNA into the assemblies. It is observed that the interactions between DODMA and RNA is slightly attractive only at low pH levels, while it becomes effectively repulsive at high and intermediate pH levels. This results into a pH dependent relocation of the RNA inside the multilayers, from the lipid head groups at low pH to a more uniform distribution inside the hydrophilic slabs of the multilayers at high pH. We observe also that at high pH levels DODMA lipids undergo a gradual shift towards the hydrophobic part of the bilayer. This results in a significant increase of the leaflet-flipping rate of DODMA, a phenomenon which may ultimately affect the fusion process of the lipoplex with the endosomal membrane. This article is protected by copyright. All rights reserved.

Keywords:  DODMA; RNA delivery; ionizable lipids; lipoplex; molecular dynamics simulations

DOI:  https://doi.org/10.1002/marc.202100683
Nat Med. 2021 Dec 09.

A phase 1 trial of lipid-encapsulated mRNA encoding a monoclonal antibody with neutralizing activity against Chikungunya virus.

Allison August, Husain Z Attarwala, Sunny Himansu, Shiva Kalidindi, Sophia Lu, Rolando Pajon, Shu Han, Jean-Michel Lecerf, Joanne E Tomassini, Marjie Hard, Leon M Ptaszek, James E Crowe, Tal Zaks.

Chikungunya virus (CHIKV) infection causes acute disease characterized by fever, rash and arthralgia, which progresses to severe and chronic arthritis in up to 50% of patients. Moreover, CHIKV infection can be fatal in infants or immunocompromised individuals and has no approved therapy or prevention. This phase 1, first-in-human, randomized, placebo-controlled, proof-of-concept trial conducted from January 2019 to June 2020 evaluated the safety and pharmacology of mRNA-1944, a lipid nanoparticle-encapsulated messenger RNA encoding the heavy and light chains of a CHIKV-specific monoclonal neutralizing antibody, CHKV-24 ( NCT03829384 ). The primary outcome was to evaluate the safety and tolerability of escalating doses of mRNA-1944 administered via intravenous infusion in healthy participants aged 18-50 years. The secondary objectives included determination of the pharmacokinetics of mRNA encoding for CHKV-24 immunoglobulin heavy and light chains and ionizable amino lipid component and the pharmacodynamics of mRNA-1944 as assessed by serum concentrations of mRNA encoding for CHKV-24 immunoglobulin G (IgG), plasma concentrations of ionizable amino lipid and serum concentrations of CHKV-24 IgG. Here we report the results of a prespecified interim analysis of 38 healthy participants who received intravenous single doses of mRNA-1944 or placebo at 0.1, 0.3 and 0.6 mg kg-1, or two weekly doses at 0.3 mg kg-1. At 12, 24 and 48 h after single infusions, dose-dependent levels of CHKV-24 IgG with neutralizing activity were observed at titers predicted to be therapeutically relevant concentrations (≥1 µg ml-1) across doses that persisted for ≥16 weeks at 0.3 and 0.6 mg kg-1 (mean t1/2 approximately 69 d). A second 0.3 mg kg-1 dose 1 week after the first increased CHKV-24 IgG levels 1.8-fold. Adverse effects were mild to moderate in severity, did not worsen with a second mRNA-1944 dose and none were serious. To our knowledge, mRNA-1944 is the first mRNA-encoded monoclonal antibody showing in vivo expression and detectable ex vivo neutralizing activity in a clinical trial and may offer a treatment option for CHIKV infection. Further evaluation of the potential therapeutic use of mRNA-1944 in clinical trials for the treatment of CHIKV infection is warranted.

DOI: https://doi.org/10.1038/s41591-021-01573-6