bims-novged 2023-02-05 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023–02–05
ten papers selected by
the Merkel lab, Ludwig-Maximilians University

Efficient Delivery of Globotriaosylceramide Synthase siRNA using Polyhistidine-Incorporated Lipid Nanoparticles.
Novel Ionizable Lipid Nanoparticles for SARS-CoV-2 Omicron mRNA Delivery.
Stat3 shRNA delivery with folate receptor-modified multi-functionalized graphene oxide particles for combined infrared radiation and gene therapy in hepatocellular carcinoma.
Targeted silencing of the MCL-1 gene using multi-layered dendrimer-based nanoconstructs achieves efficient tumor regression in xenografted mice models.
Ready-to-Use-Type Lyophilized Lipid Nanoparticle Formulation for the Postencapsulation of Messenger RNA.
Modified fibrin hydrogel for sustained delivery of RNAi lipopolyplexes in skeletal muscle.
Use of Iontophoresis Technology for Transdermal Delivery of a Minimal mRNA Vaccine as a Potential Melanoma Therapeutic.
Acyclic (S)-Glycol Nucleic Acid (S-GNA) Modification of siRNAs Improves the Safety of RNAi Therapeutics While Maintaining Potency.
Impact of the polymer backbone chemistry on interactions of amino-acid-derived zwitterionic polymers with cells.
Plasma membrane depolarization reveals endosomal escape incapacity of cell-penetrating peptides.

Macromol Biosci. 2023 Feb 02. e2200423

Efficient Delivery of Globotriaosylceramide Synthase siRNA using Polyhistidine-Incorporated Lipid Nanoparticles.

In Gyu Kim, Won Ho Jung, Gayeon You, Hyukjin Lee, Yoo Jin Shin, Sun Woo Lim, Byung Ha Chung, Hyejung Mok.

  In this study, a novel polyhistidine (pHis)-incorporated lipid nanoparticle (pHis/LNP) was developed for the delivery of therapeutic globotriaosylceramide (Gb3) synthase siRNAs using a microfluidic device with pHis as a biocompatible method of endosome escape. To inhibit the expression of Gb3 synthase, six siRNAs against Gb3 synthase were designed and an optimal siRNA sequence was selected. Selected Gb3 synthase siRNA was incorporated into pHis/LNP to prepare a spherical siRNA pHis/LNP with a size of 62.5 ± 1.9 nm and surface charge of -13.3 ± 4.2 mV. The pHis/LNP successfully protected siRNAs from degradation in 50% serum condition for 72 h. Prepared pHis/LNP exhibited superior stability for 20 days and excellent biocompatibility for A549 cells. After treatment with fluorescence-labeled LNPs, dotted fluorescent signals were co-localized with Lysotracker in cells with LNPs, whereas strong and diffused fluorescence intensity was observed in cells with pHis/LNPs probably due to successful endosomal escape. The extent of Gb3 synthase gene silencing by siRNA pHis/LNP was greatly improved (6.0-fold) compared to that by siRNA/LNP. Taken together, considering that the fabricated siRNA pHis/LNP exhibited excellent biocompatibility and superior gene silencing activity over conventional LNP, these particles can be utilized for the delivery of a wide range of therapeutic siRNAs. This article is protected by copyright. All rights reserved.

Keywords:  endosome escape; globotriaosylceramide synthase; lipid nanoparticles; polyhistidine; siRNA

DOI:  https://doi.org/10.1002/mabi.202200423
Adv Healthc Mater. 2023 Jan 30. e2202590

Novel Ionizable Lipid Nanoparticles for SARS-CoV-2 Omicron mRNA Delivery.

Jinrong Long, Changxiao Yu, Honglei Zhang, Yiming Cao, Ye Sang, Haitao Lu, Zhen Zhang, Xin Wang, Huanyu Wang, Gengshen Song, Jing Yang, Shengqi Wang.

  mRNA-based therapy has emerged as the most promising nucleic acid therapy in the fight against COVID-19. However, a safe and efficacious systemic delivery remains a challenge for mRNA therapy. Lipid nanoparticles (LNPs) are currently widely used in mRNA delivery vehicles. Here, series of ionizable LNPs are rationally designed. YK009-LNP is an optimal delivery platform to carry mRNA. YK009-LNP exhibited higher mRNA delivery efficiency, a more favorable biodistribution pattern, and better safety than the approved MC3-LNP. In addition, mRNA encoding SARS-CoV-2 Omicron receptor binding domain protein was synthesized, and intramuscular administration of mice with YK009-LNP-Omicron mRNA induced a robust immune response and immune protective effect. Our study provides a novel mRNA delivery vehicle with more powerful delivery efficiency and better safety than the approved LNPs. This article is protected by copyright. All rights reserved.

Keywords:  SARS-CoV-2 Omicron variant; ionizable lipid; lipid nanoparticles; mRNA delivery; mRNA vaccine

DOI:  https://doi.org/10.1002/adhm.202202590
Anticancer Drugs. 2022 Nov 30.

Stat3 shRNA delivery with folate receptor-modified multi-functionalized graphene oxide particles for combined infrared radiation and gene therapy in hepatocellular carcinoma.

Xuyang Chen, Ling Zhang, Xiaoqin Wang, Libo Xu, Jicheng Sun, Yiran Liu, Xiaorui Liu, Dhan V Kalvakolanu, Baofeng Guo.

As a vital oncogene, a variety of inhibitors targeting Stat3 and its various upstream signaling pathways has been explored. Since small molecules, peptidomimetics and other peptide inhibitors usually lead to side effects and difficult administration, gene therapeutics that have characteristics of low toxicity and high targeting, make them an attractive alternative for targeting Stat3. A major challenge to this approach is the lack of safe delivery systems for in-vivo applications. Among the various siRNA delivery systems, nanoparticles emerge as a new tool for gene delivery with high biocompatibility, low cost, and minimal toxicity. In this study, we developed a graphene oxide (GO)-based nanocarrier, GO-polyethyleneimine (PEI)-polyethylene glycol (PEG)-folic acid (FA), as a tool targeting for Stat3-specific shRNA to mouse hepatoma cells in vitro and in vivo. Infrared photothermal therapy was combined in vivo since GO has the characteristic of infrared absorbability. Our results suggest a significant tumor growth inhibition after treatment with GO-PEI-PEG-FA-sh-Stat3 combined with infrared photothermal therapy. Thus, GO-PEI-PEG-FA appears to be a novel nano-transformer that could be used in the clinics in future.

DOI: https://doi.org/10.1097/CAD.0000000000001461
Int J Pharm. 2023 Jan 28. pii: S0378-5173(23)00079-0. [Epub ahead of print] 122659

Targeted silencing of the MCL-1 gene using multi-layered dendrimer-based nanoconstructs achieves efficient tumor regression in xenografted mice models.

Prajakta Tambe, Rajesh Salve, Prakash Chaudhary, Pramod Kumar, Sachin Jadhav, Kishore M Paknikar, Virendra Gajbhiye.

  The setback in the practical clinical use of RNA interference (RNAi)-based cancer treatment stems from the lack of targeted small interfering RNA (siRNA) delivery. Here, we show that luteinizing hormone-releasing hormone(LHRH) analog-tethered multi-layered polyamidoamine (PAMAM) nanoconstructs silence the anti-apoptotic MCL-1 gene in LHRH receptor overexpressing human breast (MCF-7) and prostate cancer (LNCaP) cells with 70.91 % and 74.10 % efficiency, respectively. These results were confirmed by RT-PCR. The Acridine orange/Ethidium bromide (AO/EB) dual staining revealed that the silencing of MCL-1 induced apoptosis in both the cell lines. In vivo tumor regression studies performed using MCF-7 and LNCaP xenografted severe combined immunodeficiency(SCID) mice demonstrated highly improved tumor regression in groups treated with targeted nanoconstructs complexed with MCL-1 siRNA (T+siMCL-1) compared to the other treatment groups. The quantitative RT-PCR results of tumor tissues demonstrated significant MCL-1 gene silencing, i.e., 73.76% and 92.63% in breast and prostate tumors, respectively, after T+siMCL-1 treatment. Reduction in MCL-1 protein expression as assessed by immunohistochemistry further confirmed these results. Furthermore, the caspase 3/7 assay demonstrated apoptosis in the MCL-1 silenced tissues. The study strongly suggests that targeted delivery of siRNAs using multi-layered dendrimer nanostructures could be an effective therapy for LHRH overexpressing cancers.

Keywords:  Breast cancer; MCL-1; gene silencing; siRNA; tumor targeting; xenograft

DOI:  https://doi.org/10.1016/j.ijpharm.2023.122659
ACS Nano. 2023 Jan 31.

Ready-to-Use-Type Lyophilized Lipid Nanoparticle Formulation for the Postencapsulation of Messenger RNA.

Hiroki Tanaka, Shinya Hagiwara, Daiki Shirane, Takuma Yamakawa, Yuka Sato, Chika Matsumoto, Kota Ishizaki, Miho Hishinuma, Katsuyuki Chida, Kasumi Sasaki, Etsuo Yonemochi, Keisuke Ueda, Kenjirou Higashi, Kunikazu Moribe, Takashi Tadokoro, Katsumi Maenaka, Sakura Taneichi, Yuta Nakai, Kota Tange, Yu Sakurai, Hidetaka Akita.

  Based on the clinical success of an in vitro transcribed mRNA (IVT-mRNA) that is encapsulated in lipid nanoparticles (mRNA-LNPs), there is a growing demand by researchers to test whether their own biological findings might be applicable for use in mRNA-based therapeutics. However, the equipment and/or know-how required for manufacturing such nanoparticles is often inaccessible. To encourage more innovation in mRNA therapeutics, a simple method for preparing mRNA-LNPs is prerequisite. In this study, we report on a method for encapsulating IVT-mRNA into LNPs by rehydrating a Ready-to-Use empty freeze-dried LNP (LNPs(RtoU)) formulation with IVT-mRNA solution followed by heating. The resulting mRNA-LNPs(RtoU) had a similar intraparticle structure compared to the mRNA-LNPs prepared by conventional microfluidic mixing. In vivo genome editing, a promising application of these types of mRNA-LNPs, was accomplished using the LNPs(RtoU) containing co-encapsulated Cas9-mRNA and a small guide RNA.

Keywords:  freeze-drying; gene delivery; gene editing; lipid nanoparticles; mRNA delivery

DOI:  https://doi.org/10.1021/acsnano.2c10501
Regen Biomater. 2023 ;10 rbac101

Modified fibrin hydrogel for sustained delivery of RNAi lipopolyplexes in skeletal muscle.

Ellen Ngarande, Emma Doubell, Ousman Tamgue, Manuel Mano, Paul Human, Mauro Giacca, Neil Hamer Davies.

  RNA interference is a promising therapeutical approach presently hindered by delivery concerns such as rapid RNA degradation and targeting of individual tissues. Injectable hydrogels are one potentially simple and direct route towards overcoming these barriers. Here we report on the utility of a combination of a mildly modified form of the clinically utilised fibrin hydrogel with Invivofectamine® 3.0, a lipid nonviral transfection vector, for local and sustained release. PEGylation of fibrin allowed for controlled release of small interfering RNA (siRNA)-lipopolyplexes for at least 10 days and greatly increased the stability of fibrin in vitro and in vivo. A 3D cell culture model and a release study showed transfection efficacy of siRNA-lipopolyplexes was retained for a minimum of 7 days. Injection in conjunction with PEGylated-fibrinogen significantly increased retention of siRNA-lipopolyplexes in mouse skeletal muscle and enhanced knockdown of myostatin mRNA that correlated with muscle growth. Thus, the increased efficacy observed here for the combination of a lipid nanoparticle, the only type of nonviral vector approved for the clinic, with fibrin, might allow for more rapid translation of injectable hydrogel-based RNA interference.

Keywords:  RNA interference; controlled release; fibrin; lipopolyplex

DOI:  https://doi.org/10.1093/rb/rbac101
Biol Pharm Bull. 2023 ;46(2): 301-308

Use of Iontophoresis Technology for Transdermal Delivery of a Minimal mRNA Vaccine as a Potential Melanoma Therapeutic.

Rabab A Husseini, Naoko Abe, Tomoaki Hara, Hiroshi Abe, Kentaro Kogure.

  mRNA vaccines have attracted considerable attention as a result of the 2019 coronavirus pandemic; however, challenges remain regarding use of mRNA vaccines, including insufficient delivery owing to the high molecular weights and high negative charges associated with mRNA. These characteristics of mRNA vaccines impair intracellular uptake and subsequent protein translation. In the current study, we prepared a minimal mRNA vaccine encoding a tumor associated antigen human gp10025-33 peptide (KVPRNQDWL), as a potential treatment for melanoma. Minimal mRNA vaccines have recently shown promise at improving the translational process, and can be prepared via a simple production method. Moreover, we previously reported the successful use of iontophoresis (IP) technology in the delivery of hydrophilic macromolecules into skin layers, as well as intracellular delivery of small interfering RNA (siRNA). We hypothesized that combining IP technology with a newly synthesized minimal mRNA vaccine can improve both transdermal and intracellular delivery of mRNA. Following IP-induced delivery of a mRNA vaccine, an immune response is elicited resulting in activation of skin resident immune cells. As expected, combining both technologies led to potent stimulation of the immune system, which was observed via potent tumor inhibition in mice bearing melanoma. Additionally, there was an elevation in mRNA expression levels of various cytokines, mainly interferon (IFN)-γ, as well as infiltration of cytotoxic CD8+ T cells in the tumor tissue, which are responsible for tumor clearance. This is the first report demonstrating the application of IP for delivery of a minimal mRNA vaccine as a potential melanoma therapeutic.

Keywords:  iontophoresis; melanoma; minimal mRNA vaccine

DOI:  https://doi.org/10.1248/bpb.b22-00746
RNA. 2023 Feb 01. pii: rna.079526.122. [Epub ahead of print]

Acyclic (S)-Glycol Nucleic Acid (S-GNA) Modification of siRNAs Improves the Safety of RNAi Therapeutics While Maintaining Potency.

Martin Egli, Mark K Schlegel, Muthiah Manoharan.

  Glycol nucleic acid (GNA) is an acyclic nucleic acid analogue connected via phosphodiester bonds. Crystal structures of RNA-GNA chimeric duplexes indicated that nucleotides of the right-handed (S)-GNA were better accommodated in the right-handed RNA duplex than were the left-handed (R)-isomers. GNA nucleotides adopt a rotated nucleobase orientation within all duplex contexts, pairing with complementary RNA in a reverse Watson-Crick mode, which explains the inabilities of GNA C and G to form strong base pairs with complementary nucleotides. Transposition of the hydrogen bond donor and acceptor pairs using novel (S)-GNA isocytidine and isoguanosine nucleotides resulted in stable base pairing with the complementary G and C ribonucleotides, respectively. GNA nucleotide or dinucleotide incorporation into an oligonucleotide increased resistance against 3'-exonuclease-mediated degradation. Consistent with the structural observations, small interfering RNAs (siRNAs) modified with (S)-GNA had greater in vitro potencies than identical sequences containing (R)-GNA. (S)-GNA is well tolerated in the seed regions of antisense and sense strands of a GalNAc-conjugated siRNA in vitro. The siRNAs containing a GNA base pair in the seed region had in vivo potency when subcutaneously injected into mice. Importantly, seed pairing destabilization resulting from a single GNA nucleotide at position 7 of the antisense strand mitigated RNAi-mediated off-target effects in a rodent model. Two GNA-modified siRNAs have shown an improved safety profile in humans compared with their non-GNA-modified counterparts, and several additional siRNAs containing the GNA modification are currently in clinical development.

Keywords:  Glycol Nucleic Acids; Oligonucleotide Therapeutics; RNA Therapeutics; RNAi Therapeutics; siRNAs, GNA

DOI:  https://doi.org/10.1261/rna.079526.122
Bioact Mater. 2023 Jun;24 524-534

Impact of the polymer backbone chemistry on interactions of amino-acid-derived zwitterionic polymers with cells.

Meike N Leiske, Bruno G De Geest, Richard Hoogenboom.

  Zwitterionic polymers are known to interact with cells and have been shown to reveal cancer cell specificity. In this work, the importance of the chemistry of the polymer backbone for the cellular specificity of amino-acid-derived polyzwitterions is demonstrated. A series of glutamic acid (Glu)-based vinyl monomers (i.e., an acrylate, a methacrylate, an acrylamide, and a methacrylamide) were prepared and used for reversible addition-fragmentation chain-transfer (RAFT) polymerisation, yielding defined polymers with narrow size distribution (Ð < 1.3). All Glu-functionalised, zwitterionic polymers revealed high cytocompatibility; however, differences in cellular association and specificity were observed. In particular, the methacrylamide-derived polymers showed high association with both, breast cancer cells and non-cancerous dendritic cells and, consequently, lack specificity. In contrast, high specificity to only breast cancer cells was observed for polyacrylates, -methacrylates, and -acrylamides. Detailed analysis of the polymers revealed differences in hydrophobicity, zeta potential, and potential side chain hydrolysis, which are impacted by the polymer backbone and might be responsible for the altered the cell association of these polymers. It is shown that a slightly negative net charge is preferred over a neutral charge to retain cell specificity. This was also confirmed by association experiments in the presence of competitive amino acid transporter substrates. The affinity of slightly negatively charged Glu-derived polymers to the xCT Glu/cystine cell membrane antiporter was found to be higher than that of neutrally charged polymers. Our results emphasise the importance of the polymer backbone for the design of cell-specific polymers. This study further highlights the potential to tailor amino-acid-derived zwitterionic materials beyond their side chain functionality.

Keywords:  Amino-acid-functionalised polymer; Cancer-targeting; Cell-specific polymer; RAFT-Polymerisation; Zwitterionic polymer

DOI:  https://doi.org/10.1016/j.bioactmat.2023.01.005
Eur J Pharm Biopharm. 2023 Jan 26. pii: S0939-6411(23)00019-X. [Epub ahead of print]

Plasma membrane depolarization reveals endosomal escape incapacity of cell-penetrating peptides.

Marc Serulla, Palapuravan Anees, Ali Hallaj, Evgeniya Trofimenko, Tara Kalia, Yamuna Krishnan, Christian Widmann.

  Cell-penetrating peptides (CPPs) are short (<30 amino acids), generally cationic, peptides that deliver diverse cargos into cells. CPPs access the cytosol either by direct translocation through the plasma membrane or via endocytosis followed by endosomal escape. Both direct translocation and endosomal escape can occur simultaneously, making it non-trivial to specifically study endosomal escape alone. Here we depolarize the plasma membrane and showed that it inhibits the direct translocation of several CPPs but does not affect their uptake into endosomes. Despite good endocytic uptake many CPPs previously considered to access the cytosol via endosomal escape, failed to access the cytosol once direct translocation was abrogated. Even CPPs designed for enhanced endosomal escape actually showed negligible endosomal escape into the cytosol. Our data reveal that cytosolic localization of CPPs occurs mainly by direct translocation across the plasma membrane. Cell depolarization represents a simple manipulation to stringently test the endosomal escape capacity of CPPs.

Keywords:  Cell-penetrating peptides; direct translocation; endosomal escape; plasma membrane potential

DOI:  https://doi.org/10.1016/j.ejpb.2023.01.019