bims-novged 2024-03-24 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2024–03–24
eight papers selected by
the Merkel lab, Ludwig-Maximilians University

Lipid nanoparticles for local delivery of mRNA to the respiratory tract: Effect of PEG-lipid content and administration route.
A polyamino acid-based phosphatidyl polymer library for in vivo mRNA delivery with spleen targeting ability.
IL7 increases targeted lipid nanoparticle-mediated mRNA expression in T cells in vitro and in vivo by enhancing T cell protein translation.
Quantitative analysis of mRNA-lipid nanoparticle stability in human plasma and serum by size-exclusion chromatography coupled with dual-angle light scattering.
Don't shake it! Mechanical stress testing of mRNA-lipid nanoparticles.
Ionizable Lipid Nanoparticle-Mediated TRAIL mRNA Delivery in the Tumor Microenvironment to Inhibit Colon Cancer Progression.
Hybrid Poly(β-amino ester) Triblock Copolymers Utilizing a RAFT Polymerization Grafting-From Methodology.
Novel air-liquid interface culture model to investigate stiffness-dependent behaviors of alveolar epithelial cells.

Eur J Pharm Biopharm. 2024 Mar 16. pii: S0939-6411(24)00092-4. [Epub ahead of print] 114266

Lipid nanoparticles for local delivery of mRNA to the respiratory tract: Effect of PEG-lipid content and administration route.

Melike Ongun, Abhijeet Girish Lokras, Saahil Baghel, Zhenning Shi, Signe Tandrup Schmidt, Henrik Franzyk, Thomas Rades, Federica Sebastiani, Aneesh Thakur, Camilla Foged.

  Design of inhalable mRNA therapeutics is promising, because local administration in the respiratory tract is minimally invasive and induces a local response. However, several challenges related to administration via inhalation and respiratory tract barriers have so far prevented the progress of inhaled mRNA therapeutics. Here, we investigated factors of importance for lipid nanoparticle (LNP)-mediated delivery of mRNA to the respiratory tract. We hypothesized that: (i) the PEG-lipid content is important for providing colloidal stability during aerosolization and for mucosal delivery, (ii) the PEG-lipid contentinfluences mRNA expression in the lungs, and (iii) the route of administration (nasal versus pulmonary) influences mRNA expression in the lungs. In this study, we aimed to optimize the PEG-lipid content for mucosal delivery and to investigatethe effect of administration route on the kinetics of mRNA expression. Our results show that increasing the PEG-lipid content improves the colloidal stability during the aerosolization process, but has a negative impact on the transfection efficiencyin vitro. The kinetics of protein expressionin vivois dependent on the route of administration, and we found that pulmonaryadministration of mRNA-LNPs to mice results inlonger protein expression than nasaladministration. These results demonstrate that the design of the delivery system and the route of administration are importantfor achieving high mRNA transfection efficiency in the respiratory tract.

Keywords:  Lipid nanoparticle; Mucosal delivery; Nanomedicine; Pulmonary/nasal administration; Vaccine; mRNA therapeutics

DOI:  https://doi.org/10.1016/j.ejpb.2024.114266
Mater Horiz. 2024 Mar 22.

A polyamino acid-based phosphatidyl polymer library for in vivo mRNA delivery with spleen targeting ability.

Hanqin Zhao, Sheng Ma, Yibo Qi, Yuxi Gao, Yuyan Zhang, Minhui Li, Jie Chen, Wantong Song, Xuesi Chen.

A qualified delivery system is crucial for the successful application of messenger RNA (mRNA) technology. While lipid nanoparticles (LNPs) are currently the predominant platform for mRNA delivery, they encounter challenges such as high inflammation and difficulties in targeting non-liver tissues. Polymers offer a promising delivery solution, albeit with limitations including low transfection efficiency and potential high toxicity. Herein, we present a poly(L-glutamic acid)-based phosphatidyl polymeric carrier (PLG-PPs) for mRNA delivery that combines the dual advantages of phospholipids and polymers. The PLGs grafted with epoxy groups were firstly modified with different amines and then with alkylated dioxaphospholane oxides, which provided a library of PLG polymers grafted with various phosphatidyl groups. In vitro studies proved that PLG-PPs/mRNA polyplexes exhibited a significant increase in mRNA expression, peaking 14 716 times compared to their non-phosphatidyl parent polymer. Impressively, the subset PA8-PL3 not only facilitated efficient mRNA transfection but also selectively delivered mRNA to the spleen instead of the liver (resulting in 69.73% protein expression in the spleen) once intravenously administered. This type of phosphatidyl PLG polymer library provides a novel approach to the construction of mRNA delivery systems especially for spleen-targeted mRNA therapeutic delivery.

DOI: https://doi.org/10.1039/d3mh02066e
Proc Natl Acad Sci U S A. 2024 Mar 26. 121(13): e2319856121

IL7 increases targeted lipid nanoparticle-mediated mRNA expression in T cells in vitro and in vivo by enhancing T cell protein translation.

Caitlin M Tilsed, Barzan A Sadiq, Tyler E Papp, Phurin Areesawangkit, Kenji Kimura, Estela Noguera-Ortega, John Scholler, Nicholas Cerda, Haig Aghajanian, Adrian Bot, Barbara Mui, Ying Tam, Drew Weissman, Carl H June, Steven M Albelda, Hamideh Parhiz.

  The use of lipid nanoparticles (LNP) to encapsulate and deliver mRNA has become an important therapeutic advance. In addition to vaccines, LNP-mRNA can be used in many other applications. For example, targeting the LNP with anti-CD5 antibodies (CD5/tLNP) can allow for efficient delivery of mRNA payloads to T cells to express protein. As the percentage of protein expressing T cells induced by an intravenous injection of CD5/tLNP is relatively low (4-20%), our goal was to find ways to increase mRNA-induced translation efficiency. We showed that T cell activation using an anti-CD3 antibody improved protein expression after CD5/tLNP transfection in vitro but not in vivo. T cell health and activation can be increased with cytokines, therefore, using mCherry mRNA as a reporter, we found that culturing either mouse or human T cells with the cytokine IL7 significantly improved protein expression of delivered mRNA in both CD4+ and CD8+ T cells in vitro. By pre-treating mice with systemic IL7 followed by tLNP administration, we observed significantly increased mCherry protein expression by T cells in vivo. Transcriptomic analysis of mouse T cells treated with IL7 in vitro revealed enhanced genomic pathways associated with protein translation. Improved translational ability was demonstrated by showing increased levels of protein expression after electroporation with mCherry mRNA in T cells cultured in the presence of IL7, but not with IL2 or IL15. These data show that IL7 selectively increases protein translation in T cells, and this property can be used to improve expression of tLNP-delivered mRNA in vivo.

Keywords:  IL7; lipid nanoparticles; translation

DOI:  https://doi.org/10.1073/pnas.2319856121
Nanomedicine. 2024 Mar 16. pii: S1549-9634(24)00014-5. [Epub ahead of print] 102745

Quantitative analysis of mRNA-lipid nanoparticle stability in human plasma and serum by size-exclusion chromatography coupled with dual-angle light scattering.

Brian Liau, Li Zhang, Melgious Jin Yan Ang, Jian Yao Ng, C V Suresh Babu, Sonja Schneider, Ravindra Gudihal, Ki Hyun Bae, Yi Yan Yang.

  Understanding the stability of mRNA loaded lipid nanoparticles (mRNA-LNPs) is imperative for their clinical development. Herein, we propose the use of size-exclusion chromatography coupled with dual-angle light scattering (SEC-MALS) as a new approach to assessing mRNA-LNP stability in pure human serum and plasma. By applying a dual-column configuration to attenuate interference from plasma components, SEC-MALS was able to elucidate the degradation kinetics and physical property changes of mRNA-LNPs, which have not been observed accurately by conventional dynamic light scattering techniques. Interestingly, both serum and plasma had significantly different impacts on the molecular weight and radius of gyration of mRNA-LNPs, suggesting the involvement of clotting factors in desorption of lipids from mRNA-LNPs. We also discovered that a trace impurity (~1 %) in ALC-0315, identified as its O-tert-butyloxycarbonyl-protected form, greatly diminished mRNA-LNP stability in serum. These results demonstrated the potential utility of SEC-MALS for optimization and quality control of LNP formulations.

Keywords:  Dual-angle light scattering; Lipid nanoparticles; Size-exclusion chromatography; Stability; mRNA

DOI:  https://doi.org/10.1016/j.nano.2024.102745
Eur J Pharm Biopharm. 2024 Mar 14. pii: S0939-6411(24)00091-2. [Epub ahead of print] 114265

Don't shake it! Mechanical stress testing of mRNA-lipid nanoparticles.

Anna Ruppl, Denis Kiesewetter, Franziska Strütt, Monika Köll-Weber, Regine Süss, Andrea Allmendinger.

  Shaking stress studies are typically performed during formulation development to test the liability of a drug product towards interfacial stress occurring during transport, especially if a liquid formulation is desired. We evaluated various shaking procedures using a polyA-surrogate solution and verified our findings by eGFP-LNP cell-expression experiments. Shaking on an orbital shaker in vertical and horizontal orientations at increasing speeds from 300 to 600 rpm resulted in decreasing levels of encapsulated nucleic acid content, larger LNP sizes, and decreasing PDI. We report that vertical and horizontal shaking of both polyA- and eGFP-LNPs led to white deposits on the inner glass vial surface, depending on time, rpm, and temperature. Increasing the fill volume/smaller headspace (0.3 versus 0.9 mL fill) did not mitigate this phenomenon in the studied configuration, and the use of hydrophobic primary packaging even accelerated the formation of white deposits. In contrast, we demonstrated that a lyophilized polyA-LNP dosage form was less susceptible to shaking and maintained cake integrity and product properties. Multiple vortexing steps resulted in an increase in LNP size, PDI, and a decrease in encapsulated polyA content. We conclude that shaking experiments of nucleic acid-loaded LNPs in their final configuration at intended transport conditions need to be considered during technical development.

Keywords:  Formulation development; Interfacial stress; Lipid nanoparticles; Lyophilized dosage form; Nanoparticulate drug delivery systems; Nucleic acid-based therapeutics; Parenteral formulations; Shaking

DOI:  https://doi.org/10.1016/j.ejpb.2024.114265
Int J Nanomedicine. 2024 ;19 2655-2673

Ionizable Lipid Nanoparticle-Mediated TRAIL mRNA Delivery in the Tumor Microenvironment to Inhibit Colon Cancer Progression.

Walison Nunes da Silva, Pedro Augusto Carvalho Costa, Sérgio Ricardo Aluotto Scalzo Júnior, Heloísa A S Ferreira, Pedro Henrique Dias Moura Prazeres, Caroline Leonel Vasconcelos Campos, Marco Túllio Rodrigues Alves, Natália Jordana Alves da Silva, Ana Luiza de Castro Santos, Lays Cordeiro Guimarães, Maria Eduarda Chen Ferris, Ajay Thatte, Alex Hamilton, Kelly Alves Bicalho, Anderson Oliveira Lobo, Helton da Costa Santiago, Lucíola da Silva Barcelos, Maria Marta Figueiredo, Mauro Martins Teixeira, Vivian Vasconcelos Costa, Michael J Mitchell, Frédéric Frézard, Pedro Pires Goulart Guimaraes.

   Introduction: Immunotherapy has revolutionized cancer treatment by harnessing the immune system to enhance antitumor responses while minimizing off-target effects. Among the promising cancer-specific therapies, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted significant attention.
Methods: Here, we developed an ionizable lipid nanoparticle (LNP) platform to deliver TRAIL mRNA (LNP-TRAIL) directly to the tumor microenvironment (TME) to induce tumor cell death. Our LNP-TRAIL was formulated via microfluidic mixing and the induction of tumor cell death was assessed in vitro. Next, we investigated the ability of LNP-TRAIL to inhibit colon cancer progression in vivo in combination with a TME normalization approach using Losartan (Los) or angiotensin 1-7 (Ang(1-7)) to reduce vascular compression and deposition of extracellular matrix in mice.
Results: Our results demonstrated that LNP-TRAIL induced tumor cell death in vitro and effectively inhibited colon cancer progression in vivo, particularly when combined with TME normalization induced by treatment Los or Ang(1-7). In addition, potent tumor cell death as well as enhanced apoptosis and necrosis was found in the tumor tissue of a group treated with LNP-TRAIL combined with TME normalization.
Discussion: Together, our data demonstrate the potential of the LNP to deliver TRAIL mRNA to the TME and to induce tumor cell death, especially when combined with TME normalization. Therefore, these findings provide important insights for the development of novel therapeutic strategies for the immunotherapy of solid tumors.

Keywords:  TRAIL; angiotensin (1–7); immunotherapy; lipid nanoparticle; losartan; mRNA

DOI:  https://doi.org/10.2147/IJN.S452896
Macromol Chem Phys. 2023 Dec;224(24): 2300262

Hybrid Poly(β-amino ester) Triblock Copolymers Utilizing a RAFT Polymerization Grafting-From Methodology.

Karolina Kasza, Amr Elsherbeny, Cara Moloney, Kim R Hardie, Miguel Cámara, Cameron Alexander, Pratik Gurnani.

  The biocompatibility, biodegradability, and responsiveness of poly(β-amino esters) (PBAEs) has led to their widespread use as biomaterials for drug and gene delivery. Nonetheless, the step-growth polymerization mechanism that yields PBAEs limits the scope for their structural optimization toward specific applications because of limited monomer choice and end-group modifications. Moreover, to date the post-synthetic functionalization of PBAEs has relied on grafting-to approaches, challenged by the need for efficient polymer-polymer coupling and potentially difficult post-conjugation purification. Here a novel grafting-from approach to grow reversible addition-fragmentation chain transfer (RAFT) polymers from a PBAE scaffold is described. This is achieved through PBAE conversion into a macromolecular chain transfer agent through a multistep capping procedure, followed by RAFT polymerization with a range of monomers to produce PBAE-RAFT hybrid triblock copolymers. Following successful synthesis, the potential biological applications of these ABA triblock copolymers are illustrated through assembly into polymeric micelles and encapsulation of a model hydrophobic drug, followed by successful nanoparticle (NP) uptake in breast cancer cells. The findings demonstrate this novel synthetic methodology can expand the scope of PBAEs as biomaterials.

Keywords:  RAFT; Triblock copolymer; biomaterials; grafting‐from; hydrophobic drug delivery; poly(β‐amino esters)

DOI:  https://doi.org/10.1002/macp.202300262
Biochem Biophys Res Commun. 2024 Mar 15. pii: S0006-291X(24)00327-9. [Epub ahead of print]708 149791

Novel air-liquid interface culture model to investigate stiffness-dependent behaviors of alveolar epithelial cells.

Yuto Takahashi, Satoru Ito, Jungfeng Wang, Jeonghyun Kim, Takeo Matsumoto, Eijiro Maeda.

  Pulmonary alveoli are functional units in gas exchange in the lung, and their dysfunctions in lung diseases such as interstitial pneumonia are accompanied by fibrotic changes in structure, elevating the stiffness of extracellular matrix components. The present study aimed to test the hypothesis that such changes in alveoli stiffness induce functional alteration of epithelial cell functions, exacerbating lung diseases. For this, we have developed a novel method of culturing alveolar epithelial cells on polyacrylamide gel with different elastic modulus at an air-liquid interface. It was demonstrated that A549 cells on soft gels, mimicking the modulus of a healthy lung, upregulated mRNA expression and protein synthesis of surfactant protein C (SFTPC). By contrast, the cells on stiff gels, mimicking the modulus of the fibrotic lung, exhibited upregulation of SFTPC gene expression but not at the protein level. Cell morphology, as well as cell nucleus volume, were also different between the two types of gels.

Keywords:  (Up to 6); Air-liquid interface; Alveolar epithelial cells; Gel-ALI; Matrix stiffness; Polyacrylamide gel; Pulmonary fibrosis

DOI:  https://doi.org/10.1016/j.bbrc.2024.149791