bims-novged 2021-11-07 papers

on Non-viral vectors for gene delivery

Issue of 2021–11–07
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Intercalation-Driven Formation of siRNA Nanogels for Cancer Therapy.
Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells.
Quince Seed Mucilage Coated Iron Oxide Nanoparticles for Plasmid DNA Delivery.
Transient yet Robust Expression of Proteins in the Mouse Liver via Intravenous Injection of Lipid Nanoparticle-encapsulated Nucleoside-modified mRNA.
Targeted Delivery of miR-218 via Decorated Hyperbranched Polyamidoamine for Liver Cancer Regression.
Modulation of intrinsic inhibitory checkpoints using nano-carriers to unleash NK cell activity.
A Microfluidics-Based Scalable Approach to Generate Extracellular Vesicles with Enhanced Therapeutic MicroRNA Loading for Intranasal Delivery to Mouse Glioblastomas.
Genome-Editing-Mediated Restructuring of Tumor Immune Microenvironment for Prevention of Metastasis.
CRISPA: A Non-viral, Transient Cas9 Delivery System Based on Reengineered Anthrax Toxin.
A functional and self-assembling octyl-phosphonium-tagged esculetin as an effective siRNA delivery agent.
A polymer‑calcium phosphate nanocapsule for RNAi-induced oxidative stress and cascaded chemotherapy.

Nano Lett. 2021 Nov 01.

Intercalation-Driven Formation of siRNA Nanogels for Cancer Therapy.

Xiangang Huang, Gongwei Wu, Chuang Liu, Xianwu Hua, Zhongmin Tang, Yufen Xiao, Wei Chen, Jun Zhou, Na Kong, Peng Huang, Jinjun Shi, Wei Tao.

  RNA interference (RNAi) is a powerful approach in the treatment of various diseases including cancers. The clinical translation of small interfering RNA (siRNA)-based therapy requires safe and efficient delivery vehicles. Here, we report a siRNA nanogels (NG)-based delivery vehicle, which is driven directly by the intercalation between nucleic acid bis-intercalator and siRNA molecules. The intercalation-based siRNA NG exhibits good physiological stability and can enter cells efficiently via different endocytosis pathways. Furthermore, the siRNA NG can not only silence the target genes in vitro but also significantly inhibit the tumor growth in vivo. Therefore, this study provides an intercalation-based strategy for the development of a siRNA delivery platform for cancer therapy. To the best of our knowledge, this is the first report of the intercalation-driven siRNA NG.

Keywords:  RNAi; cancer therapy; intercalation; nanogels; siRNA delivery

DOI:  https://doi.org/10.1021/acs.nanolett.1c03539
Nanotechnology. 2021 Nov 02.

Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells.

Pardis Moradi, Akbar Hasanzadeh, Fatemh Radmanesh, Saideh Rajai Daryasarei, Elaheh Sadat Hosseini, Jafar Kiani, Ali Shahbazi, Helena Nourizadeh, Maryam Eslami, Akbar Dorgalaleh, Michael R Hamblin, Maryam Sahlolbei, Mahdi Karimi.

  An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with LMW PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8k in the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5 to 10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g., plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid and in vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.

Keywords:  brain; gene editing; nano-carrier; p/CRISPR transfection

DOI:  https://doi.org/10.1088/1361-6528/ac357a
Nanotechnology. 2021 Nov 02.

Quince Seed Mucilage Coated Iron Oxide Nanoparticles for Plasmid DNA Delivery.

Bahar Nikforouz, Alireza Allafchian, Seyed Amir Hossein Jalali, Hamideh Shakeripour, Rezvan Mohammadinezhad.

  This study investigates the potential of iron oxide nanoparticles (Fe3O4) and quince seed mucilage (QSM) as combined genetic carriers to deliver plasmid DNA (pDNA) through the gastrointestinal system. The samples are characterized by XRD, zeta potential, DLS, FT-IR spectroscopy, FE-SEM and VSM. The stability of pDNA loading on the nanocarriers and their release pattern are evaluated in simulated gastrointestinal environments by electrophoresis. The XRD patterns reveal that the nanocarriers could preserve their structure during various synthesis levels. The saturation magnetization (Ms) of the Fe3O4 cores are 56.48 emu/g without any magnetic hysteresis. Not only does the loaded pDNA contents experience a remarkable stability in the simulated gastric environment, but also, they could be released up to 99% when exposed to an alkaline environment similar to the intestinal fluid of fish. The results indicate that the synthesized nanoparticles could be employed as efficient low-cost pDNA carriers.

Keywords:  Electrophoresis; Gene Delivery; Iron Oxide Nanoparticles; Plasmid DNA; Quince Seed Mucilage

DOI:  https://doi.org/10.1088/1361-6528/ac3576
Bio Protoc. 2021 Oct 05. 11(19): e4184

Transient yet Robust Expression of Proteins in the Mouse Liver via Intravenous Injection of Lipid Nanoparticle-encapsulated Nucleoside-modified mRNA.

Elissa Everton, Fatima Rizvi, Anna R Smith, Mitchell Beattie, Ying Tam, Norbert Pardi, Drew Weissman, Valerie Gouon-Evans.

  With the recent availability of the SARS-CoV-2 mRNA-based vaccines, public attention has been drawn to this new technology and how it may be applied to other indications. Temporal activation of key hepatic regenerative pathways can induce liver regeneration, overcoming the lack of donor organs for liver transplantation and ineffectiveness of alternative treatments. Recombinant protein therapies and genetic therapies that target these pathways require frequent and repeated injections or, when integrated into the genome, may lead to deleterious effects. In contrast, nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA-LNP) are non-integrative and induce transient yet robust expression of proteins that could serve as an ideal therapeutic tool to treat specific liver diseases. For instance, our recent publication in Nature Communications used mRNA-LNP to express hepatic mitogens, hepatocyte growth factor, and epidermal growth factor to induce liver regeneration following both acute and chronic liver injuries. Initial testing with firefly luciferase mRNA-LNP transfection and in vivo imaging confirmed specific hepatotropic delivery. In this protocol, we describe in detail the necessary steps to deliver mRNA-LNP to the murine liver and, following intravenous injection of eGFP mRNA-LNP, verify transfection efficiency using flow cytometry and liver cell specificity using immunofluorescence analyses. This procedure presents an unprecedented tool that can be customized with mRNA-LNP encoding any protein of interest to be expressed by virtually all hepatocytes, ~70% endothelial cells, and ~40% Kupffer cells for promoting liver function and/or regeneration. Graphic abstract: Experimental Design of mRNA-LNP IV Injection and Analysis of Liver Cell Specificity and Efficiency of Transfection (Created with BioRender.com).

Keywords:  Lipid nanoparticle; Liver regeneration; Nucleoside-modified mRNA; Protein expression; Retro-orbital injection

DOI:  https://doi.org/10.21769/BioProtoc.4184
Int J Pharm. 2021 Oct 31. pii: S0378-5173(21)01062-0. [Epub ahead of print] 121256

Targeted Delivery of miR-218 via Decorated Hyperbranched Polyamidoamine for Liver Cancer Regression.

Asmaa M Elfiky, Rania Hassan Mohamed, Fatma El-Zahraa Abd El-Hakam, Mohamed A Yassin, Mahmoud ElHefnawi.

  Hepatocellular carcinoma (HCC) is one of most common causes of cancer death worldwide. MicroRNA (miRNA) replacement gene therapy is a novel approach for HCC management. MiR-218 is a promising tumor suppressor miRNA that is down-regulated in HCC. Here, our aim was the targeted delivery of miR-218 expressing DNA plasmid (pmiR-218) to suppress HCC in vitro and in vivo. Hyperbranched polyamidoamine was synthesized via simple and economically one-pot reaction followed by decoration with lactobionic acid (LA-PAMAM) to selectively deliver and restore miR-218 expression in HCC. In vitro cytotoxicity investigations revealed the high biocompatibility of LA-PAMAM. Furthermore, decoration of hyperbranched polymer with LA moieties enabled LA-PAMAM to deliver pmiR-218 more efficiently to HepG2 cells compared to both PMAMA and naked pmiR-218. Such efficient delivery of miR-218 resulted in suppression of HepG2 proliferation and down-regulation of its oncogenic HOXA1 target. In vivo, LA-PAMAM/pmiR-218 treatment of HCC induced by DEN and CCl4 in mice leads to an obvious decrease in the number and size of HCC nodules. In addition, LA-PAMAM/pmiR-218 significantly improved the liver histological features, as well as down-regulated the HOXA1 in liver tissue. In conclusion, this study showed the potential of LA-PAMAM carrier for the targeted delivery of tumor suppressor miR-218 as a therapeutic candidate for HCC.

Keywords:  Hepatocellular carcinoma; Polyamidoamine; Targeted therapy; Tumor-suppressor miRNA; miR-218

DOI:  https://doi.org/10.1016/j.ijpharm.2021.121256
EMBO Mol Med. 2021 Nov 02. e14073

Modulation of intrinsic inhibitory checkpoints using nano-carriers to unleash NK cell activity.

Guy Biber, Batel Sabag, Anat Raiff, Aviad Ben-Shmuel, Abhishek Puthenveetil, Jennifer I C Benichou, Tammir Jubany, Moria Levy, Shiran Killner, Mira Barda-Saad.

  Natural killer (NK) cells provide a powerful weapon mediating immune defense against viral infections, tumor growth, and metastatic spread. NK cells demonstrate great potential for cancer immunotherapy; they can rapidly and directly kill cancer cells in the absence of MHC-dependent antigen presentation and can initiate a robust immune response in the tumor microenvironment (TME). Nevertheless, current NK cell-based immunotherapies have several drawbacks, such as the requirement for ex vivo expansion of modified NK cells, and low transduction efficiency. Furthermore, to date, no clinical trial has demonstrated a significant benefit for NK-based therapies in patients with advanced solid tumors, mainly due to the suppressive TME. To overcome current obstacles in NK cell-based immunotherapies, we describe here a non-viral lipid nanoparticle-based delivery system that encapsulates small interfering RNAs (siRNAs) to gene silence the key intrinsic inhibitory NK cell molecules, SHP-1, Cbl-b, and c-Cbl. The nanoparticles (NPs) target NK cells in vivo, silence inhibitory checkpoint signaling molecules, and unleash NK cell activity to eliminate tumors. Thus, the novel NP-based system developed here may serve as a powerful tool for future NK cell-based therapeutic approaches.

Keywords:  Cbl; NK cells; SHP-1; immunotherapy; nanoparticles

DOI:  https://doi.org/10.15252/emmm.202114073
ACS Nano. 2021 Nov 01.

A Microfluidics-Based Scalable Approach to Generate Extracellular Vesicles with Enhanced Therapeutic MicroRNA Loading for Intranasal Delivery to Mouse Glioblastomas.

Kai Wang, Uday S Kumar, Negar Sadeghipour, Tarik F Massoud, Ramasamy Paulmurugan.

  Extracellular vesicles (EVs), including exosomes and microvesicles derived from different cell sources, are used as promising nanovesicles for delivering therapeutic microRNAs (miRNAs) and drugs in cancer therapy. However, their clinical translation is limited by the quantity, size heterogeneity, and drug or small RNA loading efficiency. Herein, we developed a scalable microfluidic platform that can load therapeutic miRNAs (antimiRNA-21 and miRNA-100) and drugs while controlling the size of microfluidically processed EVs (mpEVs) using a pressure-based disruption and reconstitution process. We prepared mpEVs of optimal size using microvesicles isolated from neural stem cells engineered to overexpress CXCR4 receptor and characterized them for charge and miRNA loading efficiency. Since the delivery of therapeutic miRNAs to brain cancer is limited by the blood-brain barrier (BBB), we adopted intranasal administration of miRNA-loaded CXCR4-engineered mpEVs in orthotopic GBM mouse models and observed a consistent pattern of mpEVs trafficking across the nasal epithelia, bypassing the BBB into the intracranial compartment. In addition, the CXCR4-engineered mpEVs manifested selective tropism toward GBMs by stromal-derived factor-1 chemotaxis to deliver their miRNA cargo. The delivered miRNAs sensitized GBM cells to temozolomide, resulting in prominent tumor regression, and improved the overall survival of mice. A simple and efficient approach of packaging miRNAs in mpEVs using microfluidics, combined with a noninvasive nose-to-brain delivery route presents far-reaching potential opportunities to improve GBM therapy in clinical practice.

Keywords:  blood brain barrier; cancer therapy; cxcr4; extracellular vesicles; microRNA; microfluidics

DOI:  https://doi.org/10.1021/acsnano.1c07587
ACS Nano. 2021 Nov 01.

Genome-Editing-Mediated Restructuring of Tumor Immune Microenvironment for Prevention of Metastasis.

Dongyoon Kim, Yina Wu, Gayong Shim, Yu-Kyoung Oh.

  Modulating the tumor immune microenvironment to activate immune cells has been investigated to convert cold to hot tumors. Here, we report that metal-lipid hybrid nanoparticle (MLN)-mediated gene editing of transforming growth factor-β (TGF-β) can restructure the tumor microenvironment to an "immune activated" state for subsequent immunotherapy. MLNs with cationic lipids and elemental metallic Au inside were designed to deliver plasmid DNA encoding TGF-β single guide RNA and Cas9 protein (pC9sTgf) and to convert near-infrared light (NIR) to heat. Upon NIR irradiation, MLNs induced photothermal anticancer effects and calreticulin exposure on B16F10 cancer cells. Lipoplexes of pC9sTgf and MLN (pC9sTgf@MLN) provided gene editing of B16F10 cells and in vivo tumor tissues. In mice treated with pC9sTgf@MLNs and NIR irradiation, the tumor microenvironment showed increases in mature dendritic cells, cytotoxic T cells, and interferon-γ expression. In B16F10 tumor-bearing mice, intratumoral injection of pC9sTgf@MLNs and NIR irradiation resulted in ablation of primary tumors. Application of pC9sTgf@MLNs and NIR irradiation prevented the growth of secondarily challenged B16F10 cells at distant sites and B16F10 lung metastasis. Combined TGF-β gene editing and phototherapy is herein supported as a modality for restructuring the tumor immune microenvironment and preventing tumor recurrence.

Keywords:  gene editing; metal−lipid hybrid nanoparticle; metastasis; transforming growth factor-β; tumor immune microenvironment

DOI:  https://doi.org/10.1021/acsnano.1c05420
Front Pharmacol. 2021 ;12 770283

CRISPA: A Non-viral, Transient Cas9 Delivery System Based on Reengineered Anthrax Toxin.

Maximilian Hirschenberger, Nicole Stadler, Maximilian Fellermann, Konstantin M J Sparrer, Frank Kirchhoff, Holger Barth, Panagiotis Papatheodorou.

  Translating the CRISPR/Cas9 genome editing technology into clinics is still hampered by rather unspecific, unsafe and/or inconvenient approaches for the delivery of its main components - the Cas9 endonuclease and a guide RNA - into cells. Here, we describe the development of a novel transient and non-viral Cas9 delivery strategy based on the translocation machinery of the Bacillus anthracis anthrax toxin, PA (protective antigen). We show that Cas9 variants fused to the N-terminus of the lethal factor or to a hexahistidine tag are shuttled through channels formed by PA into the cytosol of human cells. As proof-of-principle, we applied our new approach, denoted as CRISPA, to knock out lipolysis-stimulated lipoprotein receptor (LSR) in the human colon cancer cell line HCT116 and green-fluorescent protein (GFP) in human embryonic kidney 293T cells stably expressing GFP. Notably, we confirmed that the transporter PA can be adapted to recognize specific host cell-surface receptor proteins and may be optimized for cell type-selective delivery of Cas9. Altogether, CRISPA provides a novel, transient and non-viral way to deliver Cas9 into specific cells. Thus, this system is an additional step towards safe translation of the CRISPR/Cas9 technology into clinics.

Keywords:  CRISPR/Cas9; bacterial toxin; cell targeting; genome editing; pore-forming toxin; protein translocation; translocation channel

DOI:  https://doi.org/10.3389/fphar.2021.770283
Chem Commun (Camb). 2021 Nov 05.

A functional and self-assembling octyl-phosphonium-tagged esculetin as an effective siRNA delivery agent.

Altab Shaikh, Praveen Kumar Neeli, Gajalakshmi Singuru, Sravya Panangipalli, Rajkumar Banerjee, Sridhar Reddy Maddi, Rajamannar Thennati, Surendar Reddy Bathula, Srigiridhar Kotamraju.

Herein, we document a self-assembling octyl-TPP tagged esculetin (Mito-Esc) as functionally active and as a novel small molecule siRNA delivery vector. While Mito-Esc itself induces selective breast cancer cell death, the amphiphilic nature of Mito-Esc delivers therapeutic siRNAs intracellularly without the need for any excipient to exacerbate the anti-proliferative effects.

DOI: https://doi.org/10.1039/d1cc03497a
J Control Release. 2021 Nov 02. pii: S0168-3659(21)00583-6. [Epub ahead of print]

A polymer‑calcium phosphate nanocapsule for RNAi-induced oxidative stress and cascaded chemotherapy.

Jinsheng Huang, Chujie Zheng, Hong Xiao, Huiling Huang, Yiyao Wang, Minzhao Lin, Jun Pang, Yong Wang, Yuanyuan Yuan, Xintao Shuai.

  As most of intracellular reactive oxygen species (ROS) is produced in the mitochondria, mitochondrial modulation of cancer cell is a promising strategy for maximizing the in situ-activable combination therapy of oxidative catastrophe and cascaded chemotherapy. Herein, a serum-stable polymer‑calcium phosphate (CaP) hybrid nanocapsule carrying siRNA against ADP-ribosylation factor 6 (Arf6) overexpressed in cancer cells and parent drug camptothecin (CPT), designated as PTkCPT/siRNA, was developed for the RNAi-induced oxidative catastrophe and cascaded chemotherapy. A copolymer of mPEG-P(Asp-co-TkCPT), covalently tethered with chemotherapeutic CPT via a ROS-labile dithioketal (Tk) linker, was synthesized and self-assembled into a PTkCPT micelle as a nanotemplate for the CaP mineralization. The as-prepared PTkCPT/siRNA nanoparticle showed a core-shell-distinct nanocapsule which was consisted of a spherical polymeric core enclosed within a CaP shell capable of releasing siRNA in response to lysosomal acidity. Blocking Arf6 signal pathway of cancer cells led to their mitochondrial aggregation and subsequently induced a burst of ROS for oxidative catastrophe, which further triggered the cascaded CPT chemotherapy via the breakage of ROS-labile dithioketal linker. This strategy of RNAi-induced oxidative catastrophe and cascaded chemotherapy resulted in a significant combination effect on cancer cell killing and tumor growth inhibition in mice with low side effects, and provided a promising paradigm for precise cancer therapy.

Keywords:  Cascaded treatment; Chemotherapy; Mitochondrial modulation; Oxidative stress; Polymer prodrug; siRNA delivery

DOI:  https://doi.org/10.1016/j.jconrel.2021.10.030