bims-novged 2024-02-11 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2024–02–11
ten papers selected by
the Merkel lab, Ludwig-Maximilians University

Determination of the interior pH of lipid nanoparticles using a pH-sensitive fluorescent dye-based DNA probe.
mRNA Delivery: Challenges and Advances through Polymeric Soft Nanoparticles.
Precision treatment of viral pneumonia through macrophage-targeted lipid nanoparticle delivery.
Comparative analysis of lipid Nanoparticle-Mediated delivery of CRISPR-Cas9 RNP versus mRNA/sgRNA for gene editing in vitro and in vivo.
Substituting Poly(Ethylene Glycol) Lipids with Poly(2-Ethyl-2-Oxazoline) Lipids Improves Lipid Nanoparticle Repeat Dosing.
Enhancing gene transfection of poly(β-amino ester)s through modulation of amphiphilicity and chain sequence.
Identification of Potent siRNA Delivery Peptides Using Computer Modeling.
Characterization of mRNA Lipid Nanoparticles by Electron Density Mapping Reconstruction: X-ray Scattering with Density from Solution Scattering (DENSS) Algorithm.
Properties and synergistic effects of a nonionic backbone and aminoalkyl modified nucleosides in RNAs.
Tuning the Potency of Farnesol-Modified Polyethylenimine with Polyanionic Trans-Booster to Enhance DNA Delivery.

Biosens Bioelectron. 2024 Jan 24. pii: S0956-5663(24)00068-X. [Epub ahead of print]251 116065

Determination of the interior pH of lipid nanoparticles using a pH-sensitive fluorescent dye-based DNA probe.

Bin Zhao, Albert Kamanzi, Yao Zhang, Karen Y T Chan, Madelaine Robertson, Sabrina Leslie, Pieter R Cullis.

  Lipid nanoparticles (LNPs) containing ionizable cationic lipids are proven delivery systems for therapeutic nucleic acids, such as small interfering RNA (siRNA). It is important to understand the relationship between the interior pH of LNPs and the pH of the external environment to understand LNP formulation and function. Here, we developed a simple and rapid approach for determining the pH of the LNP core using a pH-sensitive fluorescent dye-based DNA probe. LNP siRNA systems containing pH-responsive DNA probes (LNP-siRNA&DNA) were generated by rapid mixing of lipids in ethanol and pH 4 aqueous buffer containing siRNA and DNA probes. We demonstrated that DNA probes were readily encapsulated in LNP systems and were sequestered into an environment at a high concentration as evidenced by an inter-probe FRET signal. It was shown that the pH of LNP encapsulated probes closely follows the pH increase or decrease of the external environment. This indicates that the clinically approved LNP RNA systems with similar lipid compositions (e.g., Onpattro and Comirnaty) are highly permeable to protons and that the pH of the interior environment closely mirrors the external environment. The pH-dependent response of the probe in LNPs was also confirmed under buffer conditions at various pHs. Furthermore, we showed that the pH-sensitive DNA probe can be incorporated into LNP systems at levels that allow the pH response to be monitored at a single LNP level using convex lens-induced confinement (CLiC) confocal microscopy. Direct visualization of the internal pH of single particles with the fluorescent DNA probe was achieved by CLiC for LNP-siRNA&DNA systems formulated under both high and normal ionic strength conditions.

Keywords:  Fluorescent DNA probes; Ionizable cationic lipids; Lipid nanoparticles; Single-particle imaging; pH-sensitive dye; siRNA therapeutics

DOI:  https://doi.org/10.1016/j.bios.2024.116065
Int J Mol Sci. 2024 Feb 01. pii: 1739. [Epub ahead of print]25(3):

mRNA Delivery: Challenges and Advances through Polymeric Soft Nanoparticles.

Samaneh Yousefi Adlsadabad, John W Hanrahan, Ashok Kakkar.

  Single-stranded messenger ribonucleic acid (mRNA) plays a pivotal role in transferring genetic information, and tremendous effort has been devoted over the years to utilize its transcription efficacy in therapeutic interventions for a variety of diseases with high morbidity and mortality. Lipid nanocarriers have been extensively investigated for mRNA delivery and enabled the rapid and successful development of mRNA vaccines against SARS-CoV-2. Some constraints of lipid nanocarriers have encouraged the development of alternative delivery systems, such as polymer-based soft nanoparticles, which offer a modular gene delivery platform. Such macromolecule-based nanocarriers can be synthetically articulated for tailored parameters including mRNA protection, loading efficacy, and targeted release. In this review, we highlight recent advances in the development of polymeric architectures for mRNA delivery, their limitations, and the challenges that still exist, with the aim of expediting further research and the clinical translation of such formulations.

Keywords:  cationic and non-cationic polymers; mRNA therapeutics; polymer lipid hybrid systems

DOI:  https://doi.org/10.3390/ijms25031739
Proc Natl Acad Sci U S A. 2024 Feb 13. 121(7): e2314747121

Precision treatment of viral pneumonia through macrophage-targeted lipid nanoparticle delivery.

Gan Zhao, Lulu Xue, Hannah C Geisler, Junchao Xu, Xinyuan Li, Michael J Mitchell, Andrew E Vaughan.

  Macrophages are integral components of the innate immune system, playing a dual role in host defense during infection and pathophysiological states. Macrophages contribute to immune responses and aid in combatting various infections, yet their production of abundant proinflammatory cytokines can lead to uncontrolled inflammation and worsened tissue damage. Therefore, reducing macrophage-derived proinflammatory cytokine release represents a promising approach for treating various acute and chronic inflammatory disorders. However, limited macrophage-specific delivery vehicles have hindered the development of macrophage-targeted therapies. In this study, we screened a pool of 112 lipid nanoparticles (LNPs) to identify an optimal LNP formulation for efficient siRNA delivery. Subsequently, by conjugating the macrophage-specific antibody F4/80 to the LNP surface, we constructed MacLNP, an enhanced LNP formulation designed for targeted macrophage delivery. In both in vitro and in vivo experiments, MacLNP demonstrated a significant enhancement in targeting macrophages. Specifically, delivery of siRNA targeting TAK1, a critical kinase upstream of multiple inflammatory pathways, effectively suppressed the phosphorylation/activation of NF-kB. LNP-mediated inhibition of NF-kB, a key upstream regulator in the classic inflammatory signaling pathway, in the murine macrophage cell line RAW264.7 significantly reduced the release of proinflammatory cytokines after stimulation with the viral RNA mimic Poly(I:C). Finally, intranasal administration of MacLNP-encapsulated TAK1 siRNA markedly ameliorated lung injury induced by influenza infection. In conclusion, our findings validate the potential of targeted macrophage interventions in attenuating inflammatory responses, reinforcing the potential of LNP-mediated macrophage targeting to treat pulmonary inflammatory disorders.

Keywords:  RNAi therapeutics; inhalation delivery; lipid nanoparticles; nanomedicine; pneumonia

DOI:  https://doi.org/10.1073/pnas.2314747121
Eur J Pharm Biopharm. 2024 Feb 05. pii: S0939-6411(24)00033-X. [Epub ahead of print] 114207

Comparative analysis of lipid Nanoparticle-Mediated delivery of CRISPR-Cas9 RNP versus mRNA/sgRNA for gene editing in vitro and in vivo.

Johanna Walther, Deja Porenta, Danny Wilbie, Cornelis Seinen, Naomi Benne, Qiangbing Yang, Olivier Gerrit de Jong, Zhiyong Lei, Enrico Mastrobattista.

  The discovery that the bacterial defense mechanism, CRISPR-Cas9, can be reprogrammed as a gene editing tool has revolutionized the field of gene editing. CRISPR-Cas9 can introduce a double-strand break at a specific targeted site within the genome. Subsequent intracellular repair mechanisms repair the double strand break that can either lead to gene knock-out (via the non-homologous end-joining pathway) or specific gene correction in the presence of a DNA template via homology-directed repair. With the latter, pathological mutations can be cut out and repaired. Advances are being made to utilize CRISPR-Cas9 in patients by incorporating its components into non-viral delivery vehicles that will protect them from premature degradation and deliver them to the targeted tissues. Herein, CRISPR-Cas9 can be delivered in the form of three different cargos: plasmid DNA, RNA or a ribonucleoprotein complex (RNP). We and others have recently shown that Cas9 RNP can be efficiently formulated in lipid-nanoparticles (LNP) leading to functional delivery in vitro. In this study, we compared LNP encapsulating the mRNA Cas9, sgRNA and HDR template against LNP containing Cas9-RNP and HDR template. Former showed smaller particle sizes, better protection against degrading enzymes and higher gene editing efficiencies on both reporter HEK293T cells and HEPA 1-6 cells in in vitro assays. Both formulations were additionally tested in female Ai9 mice on biodistribution and gene editing efficiency after systemic administration. LNP delivering mRNA Cas9 were retained mainly in the liver, with LNP delivering Cas9-RNPs additionally found in the spleen and lungs. Finally, gene editing in mice could only be concluded for LNP delivering mRNA Cas9 and sgRNA. These LNPs resulted in 60 % gene knock-out in hepatocytes. Delivery of mRNA Cas9 as cargo format was thereby concluded to surpass Cas9-RNP for application of CRISPR-Cas9 for gene editing in vitro and in vivo.

Keywords:  CRISPR-Cas9; Cargo format; In vitro assays; Lipid nanoparticles; Single cell flow cytometry; Systemic administration

DOI:  https://doi.org/10.1016/j.ejpb.2024.114207
Adv Healthc Mater. 2024 Feb 06. e2304033

Substituting Poly(Ethylene Glycol) Lipids with Poly(2-Ethyl-2-Oxazoline) Lipids Improves Lipid Nanoparticle Repeat Dosing.

Alejandro J Da Silva Sanchez, David Loughrey, Elisa Schrader Echeverri, Sebastian G Huayamares, Afsane Radmand, Kalina Paunovska, Marine Hatit, Karen E Tiegreen, Philip J Santangelo, James E Dahlman.

  Poly(ethylene glycol) (PEG)-lipids are used in FDA-approved lipid nanoparticle (LNP)-RNA drugs, which are safe and effective. However, it has been reported that PEG-lipids may also contribute to accelerated blood clearance and rare cases of hypersensitivity; this highlights the utility of exploring PEG-lipid alternatives. Here we show that LNPs containing poly(2-ethyl-2-oxazoline) (PEOZ)-lipids can deliver mRNA to multiple cell types in mice inside and outside the liver. In addition, we report that LNPs formulated with PEOZ-lipids show reduced clearance from the bloodstream and lower levels of anti-stealth lipid IgMs than LNPs formulated with PEG-lipids. These data justify further exploration of PEOZ-lipids as alternatives to PEG-lipids in LNP-RNA formulations. This article is protected by copyright. All rights reserved.

Keywords:  lipid nanoparticles; lnp; mrna; peg; poly(2-ethyl-2-oxazoline); polyethylene glycol

DOI:  https://doi.org/10.1002/adhm.202304033
J Control Release. 2024 Feb 06. pii: S0168-3659(24)00089-0. [Epub ahead of print]

Enhancing gene transfection of poly(β-amino ester)s through modulation of amphiphilicity and chain sequence.

Zhili Li, Rui Guo, Zhiyong Zhang, Haiyang Yong, Lei Guo, Zhengju Chen, Dongdong Huang, Dezhong Zhou.

  Poly(β-amino ester)s (PAEs) have emerged as a type of highly safe and efficient non-viral DNA delivery vectors. However, the influence of amphiphilicity and chain sequence on DNA transfection efficiency and safety profile remain largely unexplored. In this study, four PAEs with distinct amphiphilicity and chain sequences were synthesized. Results show that both amphiphilicity and chain sequence significantly affect the DNA binding and condensation ability of PAEs, as well as size, zeta potential and cellular uptake of PAE/DNA polyplexes. PAEs with different amphiphilicity and chain sequence exhibit cell type-dependent transfection capabilities: in human bladder transitional cell carcinoma (UM-UC-3), hydrophilic PAE (P-Philic) and amphiphilic PAE random copolymer (R-Amphilic) exhibit relatively higher gene transfection efficiency, while in human bladder epithelial immortalized cells (SV-HUC-1), hydrophobic PAE (P-Phobic), R-Amphilic, and amphiphilic PAE block copolymer (B-Amphilic) demonstrate higher transfection capability. Regardless of cell types, amphiphilic PAE block copolymer (B-Amphilic) always exhibits much lower gene transfection efficiency. In addition, in human colon cancer cells (HCT-116), P-Philic and R-Amphilic achieved superior gene transfection efficiency at high and low polymer/DNA weight ratios, respectively. Importantly, R-Amphilic can effectively deliver the gene encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to human chondrosarcoma cells SW1353 to induce their apoptosis, highlighting its potential application in cancer gene therapy. This study not only establishes a new paradigm for enhancing the gene transfection efficiency of PAEs by modulating their amphiphilicity and chain sequence but also identifies R-Amphilic as a potential candidate for the effective delivery of TRAIL gene in cancer gene therapy.

Keywords:  Amphiphilicity; Chain sequence; Gene delivery vectors; Gene therapy; Poly(β-amino ester)s

DOI:  https://doi.org/10.1016/j.jconrel.2024.02.002
Adv Sci (Weinh). 2024 Feb 04. e2308345

Identification of Potent siRNA Delivery Peptides Using Computer Modeling.

Ke Men, Mohan Liu, Xueyan Zhang, Yuling Yang, Rui Zhang, Yusi Wang, Die Hu, Bailing Zhou, Li Yang.

  Peptides with suitable aggregation behavior and electrical properties are potential siRNA delivery vectors. However, identifying suitable peptides with ideal delivery and safety features is difficult owing to the variations in amino acid sequences. Here, a holistic program based on computer modeling and single-cell RNA sequencing (scRNA-seq) is used to identify ideal siRNA delivery peptides. Stage one of this program consists of a sequential screening process for candidates with ideal assembly and delivery ability; stage two is a cell subtype-level analysis program that screens for high in vivo tissue safety. The leading candidate peptide selected from a library containing 12 amino acids showed strong lung-targeted siRNA delivery capacity after hydrophobic modification. Systemic administration of these compounds caused the least damage to liver and lung tissues and has little impact on macrophage and neutrophil numbers. By loading STAT3 siRNA, strong anticancer effects are achieved in multiple models, including patient-derived xenografts (PDX). This screening procedure may facilitate the development of peptide-based RNA interference (RNAi) therapeutics.

Keywords:  computer modeling; gene delivery; nanoparticle; peptide; siRNA

DOI:  https://doi.org/10.1002/advs.202308345
Pharm Res. 2024 Feb 07.

Characterization of mRNA Lipid Nanoparticles by Electron Density Mapping Reconstruction: X-ray Scattering with Density from Solution Scattering (DENSS) Algorithm.

Huy M Dao, Khaled AboulFotouh, Aasim Faheem Hussain, Alexander E Marras, Keith P Johnston, Zhengrong Cui, Robert O Williams.

   PURPOSE: This study aimed to test the feasibility of using Small Angle X-ray Scattering (SAXS) coupled with Density from Solution Scattering (DENSS) algorithm to characterize the internal architecture of messenger RNA-containing lipid nanoparticles (mRNA-LNPs).
METHODS: The DENSS algorithm was employed to construct a three-dimensional model of average individual mRNA-LNP. The reconstructed models were cross validated with cryogenic transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS) to assess size, morphology, and internal structure.
RESULTS: Cryo-TEM and DLS complemented SAXS, revealed a core-shell mRNA-LNP structure with electron-rich mRNA-rich region at the core, surrounded by lipids. The reconstructed model, utilizing the DENSS algorithm, effectively distinguishes mRNA and lipids via electron density mapping. Notably, DENSS accurately models the morphology of the mRNA-LNPs as an ellipsoidal shape with a "bleb" architecture or a two-compartment structure with contrasting electron densities, corresponding to mRNA-filled and empty lipid compartments, respectively. Finally, subtle changes in the LNP structure after three freeze-thaw cycles were detected by SAXS, demonstrating an increase in radius of gyration (Rg) associated with mRNA leakage.
CONCLUSION: Analyzing SAXS profiles based on DENSS algorithm to yield a reconstructed electron density based three-dimensional model can be a useful physicochemical characterization method in the toolbox to study mRNA-LNPs and facilitate their development.

Keywords:  DENSS; SAXS; mRNA-LNps; solution scattering

DOI:  https://doi.org/10.1007/s11095-024-03671-9
Bioorg Chem. 2024 Feb 01. pii: S0045-2068(24)00048-8. [Epub ahead of print]144 107143

Properties and synergistic effects of a nonionic backbone and aminoalkyl modified nucleosides in RNAs.

Hibiki Sawada, Yuri Kakisawa, Yoshihito Ueno.

  In this study, we report the synthesis of two formacetal (FA)-linked dimer building blocks, namely 2'-O-methyluridyl-2'-O-methyluridine and 2'-O-methyluridyl-2'-O-aminoethyluridine. We utilize the former dimer in combination with (S)-5'-C-aminopropyl-2'-O-methylnucleosides (5'-APs) as a neutral trimer unit, and the latter dimer as a cationic unit. Double-stranded RNA containing the neutral trimer unit exhibits greater stability compared to the cationic unit and maintains nuclease stability in a serum-containing buffer. Furthermore, this unit appears to establish additional hydrogen bonds with complementary bases, as supported by modeling simulations and mismatch melting temperature assays. Importantly, siRNAs modified with this unit enhance RNA interference activity in cultured cells. These findings suggest that the trimer unit holds promise for therapeutic siRNAs.

Keywords:  Aminoalkyl sidechain; Chemical modifications; RNA; SiRNA; Thermal stability

DOI:  https://doi.org/10.1016/j.bioorg.2024.107143
ACS Biomater Sci Eng. 2024 Feb 09.

Tuning the Potency of Farnesol-Modified Polyethylenimine with Polyanionic Trans-Booster to Enhance DNA Delivery.

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

  Low molecular weight polyethylenimine (PEI) based lipopolymers become an attractive strategy to construct nonviral therapeutic carriers with promising transfection efficiency and minimal toxicity. Herein, this paper presents the design and synthesis of novel farnesol (Far) conjugated PEI, namely PEI1.2k-SA-Far7. The polymers had quick DNA complexation, effective DNA unpacking (dissociation), and cellular uptake abilities when complexed with plasmid DNA. However, they were unable to provide robust transfection in culture, indicating inability of Far grafting to improve the transfection efficacy significantly. To overcome this limitation, the commercially available polyanionic Trans-Booster additive, which is capable of displaying electrostatic interaction with PEI1.2k-SA-Far7, has been used to enhance the uptake of pDNA polyplexes and transgene expression. pDNA condensation was successfully achieved in the presence of the Trans-Booster with more stable polyplexes, and in vitro transfection efficacy of the polyplexes was improved to be comparable to that obtained with an established reference reagent. The PEI1.2k-SA-Far7/pDNA/Trans-Booster ternary complex exhibited good compatibility with cells and minimal hemolysis activity. This work demonstrates the exemplary potency of using additives in polyplexes and the potential of resultant ternary complexes for effective pDNA delivery.

Keywords:  additive; gene therapy; lipopolymer; nonviral gene vector; polyethylenimine

DOI:  https://doi.org/10.1021/acsbiomaterials.4c00033