bims-novged 2023-03-05 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023‒03‒05
ten papers selected by
the Merkel lab
Ludwig-Maximilians University

Light-Activated siRNA Endosomal Release (LASER) by Porphyrin Lipid Nanoparticles.
Transfection Efficacy and Cellular Uptake of Lipid-Modified Polyethyleneimine Derivatives for Anionic Nanoparticles as Gene Delivery Vectors.
Single-cell quantification and dose-response of cytosolic siRNA delivery.
Ionizable drug delivery systems for efficient and selective gene therapy.
In utero delivery of mRNA to the heart, diaphragm and muscle with lipid nanoparticles.
Nanoparticle stereochemistry-dependent endocytic processing improves in vivo mRNA delivery.
Peptide-based siRNA delivery system for tumor vascular normalization and gene silencing in 4T1 cells.
Use of a Liver-Targeting Immune-Tolerogenic mRNA Lipid Nanoparticle Platform to Treat Peanut-Induced Anaphylaxis by Single- and Multiple-Epitope Nucleotide Sequence Delivery.
Single immunizations of self-amplifying or non-replicating mRNA-LNP vaccines control HPV-associated tumors in mice.
Toxicological Assessments of a Pandemic COVID-19 Vaccine-Demonstrating the Suitability of a Platform Approach for mRNA Vaccines.

ACS Nano. 2023 Feb 28.

Light-Activated siRNA Endosomal Release (LASER) by Porphyrin Lipid Nanoparticles.

Yulin Mo, Miffy H Y Cheng, Andrew D'Elia, Katie Doran, Lili Ding, Juan Chen, Pieter R Cullis, Gang Zheng.

  Lipid nanoparticles (LNPs) have achieved clinical success in delivering small interfering RNAs (siRNAs) for targeted gene therapy. However, endosomal escape of siRNA into the cytosol remains a fundamental challenge for LNPs. Herein, we report a strategy termed light-activated siRNA endosomal release (LASER) to address this challenge. We established a porphyrin-LNP by incorporating porphyrin-lipids into the clinically approved Onpattro formulation. The porphyrin-LNP maintained the physical properties of an LNP and generated reactive oxygen species (ROS) when irradiated with near-infrared (NIR) light. Using confocal microscopy, we revealed that porphyrin-lipids within the LNP translocate to endosomal membranes during endocytosis. The translocated porphyrin-lipids generated ROS under light irradiation and enabled LASER through endosomal membranes disruption as observed through GAL-9 recruitment and transmission electron microscopy (TEM). By establishing a quantitative confocal imaging method, we confirmed that porphyrin-LNPs can increase siRNA endosomal escape efficiency by up to 2-fold via LASER and further enhance luciferase target knockdown by 4-fold more in luciferase-transfected prostate cancer cells. Finally, we formulated porphyrin-LNPs encapsulated with gold nanoparticles (GNP) and visualized the LASER effect within prostate tumors via TEM, confirming the light-activated endosomal membrane disruption and subsequent GNP release into cytosols in vivo. Overall, porphyrin-LNPs and the LASER approach enhanced siRNA endosomal escape and significantly improved knockdown efficacy. We believe the versatility of this technology could be applied to various LNP-based RNA therapeutics.

Keywords:  RNA delivery; endosomal escape; lipid nanoparticles; nanomedicine; photochemical internalization; photosensitizer; porphyrin lipid

DOI:  https://doi.org/10.1021/acsnano.2c10936
ACS Appl Bio Mater. 2023 Feb 28.

Transfection Efficacy and Cellular Uptake of Lipid-Modified Polyethyleneimine Derivatives for Anionic Nanoparticles as Gene Delivery Vectors.

Amarnath Praphakar Rajendran, Oluwanifemi Ogundana, Luis Carlos Morales, Daniel Nisakar Meenakshi Sundaram, Cezary Kucharski, Remant Kc, Hasan Uludağ.

  Cationic polyethylenimine (PEI)-based nonviral gene carriers have been desirable to overcome the limitations of viral vectors in gene therapy. A range of PEI derivatives were designed, synthesized, and evaluated for nonviral delivery applications of plasmid DNA (pDNA). Linolenic acid, lauric acid, and oleic acid were covalently conjugated with low-molecular-weight PEI (Mw ∼ 1200 Da) via two different linkers, gallic acid (GA) and p-hydroxybenzoic acid (PHPA), that allows a differential loading of lipids per modified amine (3 vs 1, respectively). 1H NMR spectrum confirmed the expected structure of the conjugates as well as the level of lipid substitution. SYBR Green binding assay performed to investigate the 50% binding concentration (BC50) of lipophilic polymers to pDNA revealed increased BC50 with an increased level of lipid substitution. The particle analysis determined that GA- and PHPA-modified lipopolymers gave pDNA complexes with ∼300 and ∼100 nm in size, respectively. At the polymer/pDNA ratio of 5.0, the ζ-potentials of the complexes were negative (-6.55 to -10.6 mV) unlike the complexes with the native PEI (+11.2 mV). The transfection experiments indicated that the prepared lipopolymers showed higher transfection in attachment-dependent cells than in suspension cells based on the expression of the reporter green fluorescent protein (GFP) gene. When loaded with Cy3-labeled pDNA, the lipopolymers exhibited effective cellular uptake in attachment-dependent cells while the cellular uptake was limited in suspension cells. These results demonstrate the potential of lipid-conjugated PEI via GA and PHPA linkers, which are promising for the modification of anchorage-dependent cells.

Keywords:  gene therapy; lipophilic polymers; nonviral delivery; polyethylenimine; transfection

DOI:  https://doi.org/10.1021/acsabm.2c00978
Nat Commun. 2023 Feb 25. 14(1): 1075

Single-cell quantification and dose-response of cytosolic siRNA delivery.

Hampus Hedlund, Hampus Du Rietz, Johanna M Johansson, Hanna C Eriksson, Wahed Zedan, Linfeng Huang, Jonas Wallin, Anders Wittrup.

Endosomal escape and subsequent cytosolic delivery of small interfering RNA (siRNA) therapeutics is believed to be highly inefficient. Since it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determining delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to quantify cytosolic delivery of fluorescently labeled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP unveiled a dose-response relationship with respect to knockdown induction, depth and duration in the range from several hundred to thousands of cytosolic siRNA molecules. Accurate quantification of cytosolic siRNA, and the establishment of the intracellular dose-response relationships, will aid the development and characterization of novel delivery strategies for nucleic acid therapeutics.

DOI: https://doi.org/10.1038/s41467-023-36752-1
Mil Med Res. 2023 Feb 27. 10(1): 9

Ionizable drug delivery systems for efficient and selective gene therapy.

Yu-Qi Zhang, Ran-Ran Guo, Yong-Hu Chen, Tian-Cheng Li, Wen-Zhen Du, Rong-Wu Xiang, Ji-Bin Guan, Yu-Peng Li, Yuan-Yu Huang, Zhi-Qiang Yu, Yin Cai, Peng Zhang, Gui-Xia Ling.

  Gene therapy has shown great potential to treat various diseases by repairing the abnormal gene function. However, a great challenge in bringing the nucleic acid formulations to the market is the safe and effective delivery to the specific tissues and cells. To be excited, the development of ionizable drug delivery systems (IDDSs) has promoted a great breakthrough as evidenced by the approval of the BNT162b2 vaccine for prevention of coronavirus disease 2019 (COVID-19) in 2021. Compared with conventional cationic gene vectors, IDDSs can decrease the toxicity of carriers to cell membranes, and increase cellular uptake and endosomal escape of nucleic acids by their unique pH-responsive structures. Despite the progress, there remain necessary requirements for designing more efficient IDDSs for precise gene therapy. Herein, we systematically classify the IDDSs and summarize the characteristics and advantages of IDDSs in order to explore the underlying design mechanisms. The delivery mechanisms and therapeutic applications of IDDSs are comprehensively reviewed for the delivery of pDNA and four kinds of RNA. In particular, organ selecting considerations and high-throughput screening are highlighted to explore efficiently multifunctional ionizable nanomaterials with superior gene delivery capacity. We anticipate providing references for researchers to rationally design more efficient and accurate targeted gene delivery systems in the future, and indicate ideas for developing next generation gene vectors.

Keywords:  Gene therapy; Ionizable drug delivery systems (IDDSs); Ionizable nanomaterials; Nucleic acids

DOI:  https://doi.org/10.1186/s40779-023-00445-z
Bioact Mater. 2023 Jul;25 387-398

In utero delivery of mRNA to the heart, diaphragm and muscle with lipid nanoparticles.

Kewa Gao, Jie Li, Hengyue Song, Hesong Han, Yongheng Wang, Boyan Yin, Diana L Farmer, Niren Murthy, Aijun Wang.

  Nanoparticle-based drug delivery systems have the potential to revolutionize medicine, but their low vascular permeability and rapid clearance by phagocytic cells have limited their medical impact. Nanoparticles delivered at the in utero stage can overcome these key limitations due to the high rate of angiogenesis and cell division in fetal tissue and the under-developed immune system. However, very little is known about nanoparticle drug delivery at the fetal stage of development. In this report, using Ai9 CRE reporter mice, we demonstrate that lipid nanoparticle (LNP) mRNA complexes can deliver mRNA in utero, and can access and transfect major organs, such as the heart, the liver, kidneys, lungs and the gastrointestinal tract with remarkable efficiency and low toxicity. In addition, at 4 weeks after birth, we demonstrate that 50.99 ± 5.05%, 36.62 ± 3.42% and 23.7 ± 3.21% of myofiber in the diaphragm, heart and skeletal muscle, respectively, were transfected. Finally, we show here that Cas9 mRNA and sgRNA complexed to LNPs were able to edit the fetal organs in utero. These experiments demonstrate the possibility of non-viral delivery of mRNA to organs outside of the liver in utero, which provides a promising strategy for treating a wide variety of devastating diseases before birth.

Keywords:  CRISPR/Cas9; Gene editing; In utero; Nanoparticles; mRNA delivery

DOI:  https://doi.org/10.1016/j.bioactmat.2023.02.011
Nat Chem. 2023 Mar 02.

Nanoparticle stereochemistry-dependent endocytic processing improves in vivo mRNA delivery.

Marine Z C Hatit, Curtis N Dobrowolski, Melissa P Lokugamage, David Loughrey, Huanzhen Ni, Chiara Zurla, Alejandro J Da Silva Sanchez, Afsane Radmand, Sebastian G Huayamares, Ryan Zenhausern, Kalina Paunovska, Hannah E Peck, Jinwhan Kim, Manaka Sato, Jacob I Feldman, Michael-Alexander Rivera, Ana Cristian, YongTae Kim, Philip J Santangelo, James E Dahlman.

Stereochemistry can alter small-molecule pharmacokinetics, safety and efficacy. However, it is unclear whether the stereochemistry of a single compound within a multicomponent colloid such as a lipid nanoparticle (LNP) can influence its activity in vivo. Here we report that LNPs containing stereopure 20α-hydroxycholesterol (20α) delivered mRNA to liver cells up to 3-fold more potently than LNPs containing a mixture of both 20α- and 20β-hydroxycholesterols (20mix). This effect was not driven by LNP physiochemical traits. Instead, in vivo single-cell RNA sequencing and imaging revealed that 20mix LNPs were sorted into phagocytic pathways more than 20α LNPs, resulting in key differences between LNP biodistribution and subsequent LNP functional delivery. These data are consistent with the fact that nanoparticle biodistribution is necessary, but not sufficient, for mRNA delivery, and that stereochemistry-dependent interactions between LNPs and target cells can improve mRNA delivery.

DOI: https://doi.org/10.1038/s41557-023-01138-9
STAR Protoc. 2023 Feb 28. pii: S2666-1667(23)00096-5. [Epub ahead of print]4(1): 102138

Peptide-based siRNA delivery system for tumor vascular normalization and gene silencing in 4T1 cells.

Yunfei Yi, Chan Feng, Mian Yu, Lin Mei, Meiying Wu, Wei Tao.

  Efficient gene delivery in an integrated drug delivery system is urgent for multimodal antitumor therapy. Herein, we describe a protocol for constructing a peptide-based siRNA delivery system to achieve tumor vascular normalization and gene silencing in 4T1 cells. We highlighted four major steps, including (1) synthesis of the chimeric peptide, (2) preparation and characterization of PA7R@siRNA micelleplexes, (3) in vitro tube formation assay and transwell cell migration assay, and (4) siRNA transfection in 4T1 cells. This delivery system is expected to be used to silence gene expression, normalize tumor vasculature, and perform other treatments based on the different peptide segments. For complete details on the use and execution of this protocol, please refer to Yi et al. (2022).1.

Keywords:  Biotechnology and Bioengineering; Cancer; Chemistry; Health Sciences; Molecular Biology

DOI:  https://doi.org/10.1016/j.xpro.2023.102138
ACS Nano. 2023 Feb 28.

Use of a Liver-Targeting Immune-Tolerogenic mRNA Lipid Nanoparticle Platform to Treat Peanut-Induced Anaphylaxis by Single- and Multiple-Epitope Nucleotide Sequence Delivery.

Xiao Xu, Xiang Wang, Yu-Pei Liao, Lijia Luo, Tian Xia, Andre E Nel.

  While oral desensitization is capable of alleviating peanut allergen anaphylaxis, long-term immune tolerance is the sought-after goal. We developed a liver-targeting lipid nanoparticle (LNP) platform to deliver mRNA-encoded peanut allergen epitopes to liver sinusoidal endothelial cells (LSECs), which function as robust tolerogenic antigen-presenting cells that induce FoxP3+ regulatory T-cells (Tregs). The mRNA strand was constructed by including nucleotide sequences encoding for nonallergenic MHC-II binding T-cell epitopes, identified in the dominant peanut allergen, Ara h2. These epitopes were inserted in the mRNA strand downstream of an MHC-II targeting sequence, further endowed in vitro with 5' and 3' capping sequences, a PolyA tail, and uridine substitution. Codon-optimized mRNA was used for microfluidics synthesis of LNPs with an ionizable cationic lipid, also decorated with a lipid-anchored mannose ligand for LSEC targeting. Biodistribution to the liver was confirmed by in vivo imaging, while ELISpot assays demonstrated an increase in IL-10-producing Tregs in the spleen. Prophylactic administration of tandem-repeat or a combination of encapsulated Ara h2 epitopes induced robust tolerogenic effects in C3H/HeJ mice, sensitized to and subsequently challenged with crude peanut allergen extract. In addition to alleviating physical manifestations of anaphylaxis, there was suppression of Th2-mediated cytokine production, IgE synthesis, and mast cell release, accompanied by increased IL-10 and TGF-β production in the peritoneum. Similar efficacy was demonstrated during LNP administration postsensitization. While nondecorated particles had lesser but significant effects, PolyA/LNP-Man lacked protective effects. These results demonstrate an exciting application of mRNA/LNP for treatment of food allergen anaphylaxis, with the promise to be widely applicable to the allergy field.

Keywords:  anaphylaxis; lipid nanoparticles; liver-targeting; mRNA delivery; peanut allergy

DOI:  https://doi.org/10.1021/acsnano.2c12420
Sci Transl Med. 2023 Mar 03. eabn3464

Single immunizations of self-amplifying or non-replicating mRNA-LNP vaccines control HPV-associated tumors in mice.

Jamile Ramos da Silva, Karine Bitencourt Rodrigues, Guilherme Formoso Pelegrin, Natiely Silva Sales, Hiromi Muramatsu, Mariângela de Oliveira Silva, Bruna F M M Porchia, Ana Carolina Ramos Moreno, Luana Raposo M M Aps, Aléxia Adrianne Venceslau-Carvalho, István Tombácz, Wesley Luzetti Fotoran, Katalin Karikó, Paulo J C Lin, Ying K Tam, Mariana de Oliveira Diniz, Norbert Pardi, Luís Carlos de Souza Ferreira.

As mRNA vaccines have proved to be very successful in battling the coronavirus disease 2019 (COVID-19) pandemic, this new modality has attracted widespread interest for the development of potent vaccines against other infectious diseases and cancer. Cervical cancer caused by persistent human papillomavirus (HPV) infection is a major cause of cancer-related deaths in women, and the development of safe and effective therapeutic strategies is urgently needed. In the present study, we compared the performance of three different mRNA vaccine modalities to target tumors associated with HPV-16 infection in mice. We generated lipid nanoparticle (LNP)-encapsulated self-amplifying mRNA as well as unmodified and nucleoside-modified non-replicating mRNA vaccines encoding a chimeric protein derived from the fusion of the HPV-16 E7 oncoprotein and the herpes simplex virus type 1 glycoprotein D (gDE7). We demonstrated that single low-dose immunizations with any of the three gDE7 mRNA vaccines induced activation of E7-specific CD8+ T cells, generated memory T cell responses capable of preventing tumor relapses, and eradicated subcutaneous tumors at different growth stages. In addition, the gDE7 mRNA-LNP vaccines induced potent tumor protection in two different orthotopic mouse tumor models after administration of a single vaccine dose. Last, comparative studies demonstrated that all three gDE7 mRNA-LNP vaccines proved to be superior to gDE7 DNA and gDE7 recombinant protein vaccines. Collectively, we demonstrated the immunogenicity and therapeutic efficacy of three different mRNA vaccines in extensive comparative experiments. Our data support further evaluation of these mRNA vaccines in clinical trials.

DOI: https://doi.org/10.1126/scitranslmed.abn3464
Vaccines (Basel). 2023 Feb 11. pii: 417. [Epub ahead of print]11(2):

Toxicological Assessments of a Pandemic COVID-19 Vaccine-Demonstrating the Suitability of a Platform Approach for mRNA Vaccines.

Cynthia M Rohde, Claudia Lindemann, Michael Giovanelli, Rani S Sellers, Jan Diekmann, Shambhunath Choudhary, Lila Ramaiah, Annette B Vogel, Yana Chervona, Alexander Muik, Ugur Sahin.

  The emergence of SARS-CoV-2 at the end of 2019 required the swift development of a vaccine to address the pandemic. Nonclinical GLP-compliant studies in Wistar Han rats were initiated to assess the local tolerance, systemic toxicity, and immune response to four mRNA vaccine candidates encoding immunogens derived from the spike (S) glycoprotein of SARS-CoV-2, encapsulated in lipid nanoparticles (LNPs). Vaccine candidates were administered intramuscularly once weekly for three doses at 30 and/or 100 µg followed by a 3-week recovery period. Clinical pathology findings included higher white blood cell counts and acute phase reactant concentrations, lower platelet and reticulocyte counts, and lower RBC parameters. Microscopically, there was increased cellularity (lymphocytes) in the lymph nodes and spleen, increased hematopoiesis in the bone marrow and spleen, acute inflammation and edema at the injection site, and minimal hepatocellular vacuolation. These findings were generally attributed to the anticipated immune and inflammatory responses to the vaccines, except for hepatocyte vacuolation, which was interpreted to reflect hepatocyte LNP lipid uptake, was similar between candidates and resolved or partially recovered at the end of the recovery phase. These studies demonstrated safety and tolerability in rats, supporting SARS-CoV-2 mRNA-LNP vaccine clinical development.

Keywords:  BNT162b1; BNT162b2; BNT162b3; COVID-19 mRNA vaccine; nonclinical safety; rat; toxicity

DOI:  https://doi.org/10.3390/vaccines11020417