bims-novged 2022-12-25 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2022–12–25
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University

A Polymeric Nanoparticle Formulation for Targeted mRNA Delivery to Fibroblasts.
Cationic lipid modification of DNA tetrahedral nanocages enhances their cellular uptake.
Gene transfection using branched cationic amphiphilic compounds for an aerosol administration in cystic fibrosis context.
Lipid nanoparticle-based mRNA candidates elicit potent T cell responses.
Fluoroalkane modified cationic polymers for personalized mRNA cancer vaccines.
A Multifunctional Nano-Delivery System Against Rheumatoid Arthritis by Combined Phototherapy, Hypoxia-Activated Chemotherapy, and RNA Interference.
Analysis of polymeric nanoparticle properties for siRNA/DNA delivery in a tumor xenograft tissue slice air-liquid interface model.
Delivering mRNA to Secondary Lymphoid Tissues by Phosphatidylserine-loaded Lipid Nanoparticles.
Nanoparticle delivery of CD40 siRNA suppresses alloimmune responses by inhibiting activation and differentiation of DCs and macrophages.
Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins.
Manganese-coordinated mRNA vaccines with enhanced mRNA expression and immunogenicity induce robust immune responses against SARS-CoV-2 variants.

Adv Sci (Weinh). 2022 Dec 18. e2205475

A Polymeric Nanoparticle Formulation for Targeted mRNA Delivery to Fibroblasts.

Artur Filipe Rodrigues, Catarina Rebelo, Susana Simões, Cristiana Paulo, Sónia Pinho, Vítor Francisco, Lino Ferreira.

  Messenger RNA (mRNA)-based therapies offer enhanced control over the production of therapeutic proteins for many diseases. Their clinical implementation warrants formulations capable of delivering them safely and effectively to target sites. Owing to their chemical versatility, polymeric nanoparticles can be designed by combinatorial synthesis of different ionizable, cationic, and aromatic moieties to modulate cell targeting, using inexpensive formulation steps. Herein, 152 formulations are evaluated by high-throughput screening using a reporter fibroblast model sensitive to functional delivery of mRNA encoding Cre recombinase. Using in vitro and in vivo models, a polymeric nanoformulation based on the combination of 3 specific monomers is identified to transfect fibroblasts much more effectively than other cell types populating the skin, with superior performance than lipid-based transfection agents in the delivery of Cas9 mRNA and guide RNA. This tropism can be explained by receptor-mediated endocytosis, involving CD26 and FAP, which are overexpressed in profibrotic fibroblasts. Structure-activity analysis reveals that efficient mRNA delivery required the combination of high buffering capacity and low mRNA binding affinity for rapid release upon endosomal escape. These results highlight the use of high-throughput screening to rapidly identify chemical features towards the design of highly efficient mRNA delivery systems targeting fibrotic diseases.

Keywords:  CRISPR/Cas9; gene edition; high-throughput screening; messenger RNA; polymeric nanoparticles

DOI:  https://doi.org/10.1002/advs.202205475
Nanoscale. 2022 Dec 23.

Cationic lipid modification of DNA tetrahedral nanocages enhances their cellular uptake.

Ramesh Singh, Pankaj Yadav, Hema Naveena A, Dhiraj Bhatia.

Self-assembled DNA nanocages are among the most promising candidates for bioimaging and payload delivery into cells. DNA nanocages have great potential to efficiently address drug resistance and nucleic acid delivery problems due to precise control of their shape and size, and excellent biocompatibility. Although DNA nanostructures demonstrate some cellular uptake, because they bear a highly negative charge, the uptake of tetrahedral nanostructures is hindered by electrostatic repulsion. In this study, we describe a method to enhance the cellular uptake of DNA nanostructures using a binary system containing DNA and a positively charged head group with a hydrophobic lipid chain containing lipids for cellular internalization. Here we represent the functionalization of a model cage, DNA tetrahedron (TD) with a cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Atomic force microscopy (AFM) and other standard characterization techniques were used to explore the co-assembly of the DNA tetrahedron and DOTMA. We revealed a simple confocal microscopy-based approach to show the enhancement in the cellular uptake of DNA nanocages. This new method will find multiple applications in delivery applications such as gene transfection, drug delivery and targeted bioimaging.

DOI: https://doi.org/10.1039/d2nr05749b
Int J Pharm. 2022 Dec 15. pii: S0378-5173(22)01046-8. [Epub ahead of print]631 122491

Gene transfection using branched cationic amphiphilic compounds for an aerosol administration in cystic fibrosis context.

Rosy Ghanem, Mathieu Berchel, Tanguy Haute, Xavier Buin, Véronique Laurent, Raphaëlle Youf, Amal Bouraoui, Tony Le Gall, Paul-Alain Jaffrès, Tristan Montier.

  For cystic fibrosis gene therapy, the aerosolization of genetic materials is the most relevant delivery strategy to reach the airway epithelium. However, aerosolized formulations have to resist shear forces while maintaining the integrity of plasmid DNA (pDNA) during its journey from the nebulization to the epithelial cells. Herein, we compared the efficiency of gene delivery by aerosolization of two types of formulations: (i) BSV163, a branched cationic amphiphilic compound, co-formulated with different DOPE ratios (mol/mol) and DMPE-PEG5000 and (ii) 25 KDa branched polyethylenimine (b-PEI)-based formulation used as control. This study also aims to determine whether BSV163-based formulations possess the ability to resist the nebulization mechanisms and protect the nucleic acids (pDNA) cargo. Therefore, two CpG free plasmids (pGM144 or pGM169) encoding either the luciferase reporter gene or hCFTR respectively were used. Air-Liquid Interface (ALI) cell-culture was selected as an in-vitro model for aerosol experiments due to its closer analogy with in vivo morphology. Results highlighted that DOPE ratio influences the capacity of the BSV163 based-formulations to mediate high transfection efficacies. Furthermore, we proved that addition of DMPE-PEG5000 upon the formation of the BSV163/DOPE (1/1) lipid film instead of post-insertion led to a higher transgene expression. The aerosolization of this formulation on ALI cell-culture was more efficient than the use of b-PEI-based formulation.

Keywords:  Aerosol administration; Amphiphilic compounds; Cystic Fibrosis; Non-viral gene delivery

DOI:  https://doi.org/10.1016/j.ijpharm.2022.122491
Biomater Sci. 2022 Dec 20.

Lipid nanoparticle-based mRNA candidates elicit potent T cell responses.

Ye Zeng, Oscar Escalona-Rayo, Renzo Knol, Alexander Kros, Bram Slütter.

The induction of a potent T cell response is essential for successful tumor immunotherapy and protection against many infectious diseases. In the past few years, mRNA vaccines have emerged as potent immune activators and inducers of a robust T cell immune response. The recent approval of the Moderna and the Pfizer/BioNTech vaccines based on lipid nanoparticles (LNP) encapsulating antigen-encoding mRNA has revolutionized the field of vaccines. The advantages of LNPs are their ease of design and formulation resulting in potent, effective, and safe vaccines. However, there is still plenty of room for improvement with respect to LNP efficacy, for instance, by optimizing the lipid composition and tuning LNP for specific purposes. mRNA delivery is known to be strongly dependent on the lipid composition of LNPs and the efficiency is mainly determined by the ionizable lipids. Besides that, cholesterol and helper lipids also play important roles in mRNA transfection potency. Here, a panel of LNP formulations was studied by keeping the ionizable lipids constant, replacing cholesterol with β-sitosterol, and changing the fusogenic helper lipid DOPE content. We studied the ability of this LNP library to induce antigen presentation and T cell proliferation to identify superior LNP candidates eliciting potent T cell immune responses. We hypothesize that using β-sitosterol and increasing DOPE content would boost the mRNA transfection on immune cells and result in enhanced immune responses. Transfection of immortal immune cell lines and bone marrow dendritic cells (BMDCs) with LNPs was studied. Delivery of mRNA coding for the model antigen ovalbumin (OVA-mRNA) to BMDCs with a number of LNP formulations, resulted in a high level of activation, as evidenced by the upregulation of the co-stimulatory receptors (CD40 and CD86) and IL-12 in BMDCs. The enhancement of BMDC activation and T cell proliferation induced by the introduction of β-sitosterol and fusogenic DOPE lipids were cell dependent. Four LNP formulations (C12-200-cho-10%DOPE, C12-200-sito-10%DOPE, cKK-E12-cho-10%DOPE and cKK-E12-sito-30%DOPE) were identified that induced robust T cell proliferation and enhanced IFN-γ, TNF-α, IL-2 expression. These results demonstrate that T cell proliferation is strongly dependent on LNP composition and promising LNP-mRNA vaccine formulations were identified.

DOI: https://doi.org/10.1039/d2bm01581a
Chem Eng J. 2023 Jan 15. 456 140930

Fluoroalkane modified cationic polymers for personalized mRNA cancer vaccines.

Junyan Li, Yuanyuan Wu, Jian Xiang, Hairong Wang, Qi Zhuang, Ting Wei, Zhiqin Cao, Qingyang Gu, Zhuang Liu, Rui Peng.

  Messenger RNA (mRNA) vaccines, while demonstrating great successes in the fight against COVID-19, have been extensively studied in other areas such as personalized cancer immunotherapy based on tumor neoantigens. In addition to the design of mRNA sequences and modifications, the delivery carriers are also critical in the development of mRNA vaccines. In this work, we synthesized fluoroalkane-grafted polyethylenimine (F-PEI) for mRNA delivery. Such F-PEI could promote intracellular delivery of mRNA and activate the Toll-like receptor 4 (TLR4)-mediated signaling pathway. The nanovaccine formed by self-assembly of F-PEI and the tumor antigen-encoding mRNA, without additional adjuvants, could induce the maturation of dendritic cells (DCs) and trigger efficient antigen presentation, thereby eliciting anti-tumor immune responses. Using the mRNA encoding the model antigen ovalbumin (mRNAOVA), our F-PEI-based mRNAOVA cancer vaccine could delay the growth of established B16-OVA melanoma. When combined with immune checkpoint blockade therapy, the F-PEI-based MC38 neoantigen mRNA cancer vaccine was able to suppress established MC38 colon cancer and prevent tumor reoccurrence. Our work presents a new tool for mRNA delivery, promising not only for personalized cancer vaccines but also for other mRNA-based immunotherapies.

Keywords:  Antigen presentation; Cancer immunotherapy; Fluoropolymer; Personalized mRNA cancer vaccine; mRNA Delivery

DOI:  https://doi.org/10.1016/j.cej.2022.140930
Int J Nanomedicine. 2022 ;17 6257-6273

A Multifunctional Nano-Delivery System Against Rheumatoid Arthritis by Combined Phototherapy, Hypoxia-Activated Chemotherapy, and RNA Interference.

Xiangyu Li, Shixin Zhang, Miaomiao Zhang, Ge Li, Bo Yang, Xinyue Lu, Lesheng Teng, Youxin Li, Fengying Sun.

   Purpose: Effective therapy for rheumatoid arthritis (RA) keeps a challenge due to the complex pathogenesis of RA. It is not enough to completely inhibit the process of RA with any single therapy method. The purpose of the research is to compensate for the insufficiency of monotherapy using multiple treatment regimens with different mechanisms.
Material and Methods: In this study, we developed a new method to synthesize mesoporous silica nanoparticles hybridized with photosensitizer PCPDTBT (HNs). Branched polyethyleneimine-folic acid (PEI-FA) could be coated on the surface of HNs through electrostatic interactions. It simultaneously blocked the hypoxia-activated prodrug tirapazamine loaded into the mesopores and binded with Mcl-1 siRNA (siMcl-1) that interfered with the expression of the anti-apoptotic protein Mcl-1. Released from the co-delivery nanoparticles (PFHNs/TM) Tirapazamine and siMcl-1 upon exposure to acidic conditions of endosomes/lysosomes in activated macrophages. Under NIR irradiation, photothermal therapy and photodynamic therapy derived from PCPDTBT, hypoxia-activated chemotherapy derived from tirapazamine, and RNAi derived from siMcl-1 were used for the combined treatment for RA by killing activated macrophages. PEI-FA-coated PFHNs/TM exhibited activated macrophage-targeting characteristics, thereby enhancing the in vitro and in vivo NIR-induced combined treatment of RA.
Results: The prepared PFHNs/TM have high blood compatibility (far below 5% of hemolysis) and ideal in vitro phototherapy effect while controlling the TPZ release and binding siMcl-1. We prove that PEI-FA-coated PFHNs/TM not only protect the bound siRNA but also are selectively uptaked by activated macrophages through FA receptor-ligand-mediated endocytosis, and effectively silence the target anti-apoptotic protein by siMcl-1 transfection. In vivo, PFHNs/TM have also been revealed to be selectively enriched at the inflammatory site of RA, exhibiting NIR-induced anti-RA efficacy.
Conclusion: Overall, these FA-functionalized, pH-responsive PFHNs/TM represent a promising platform for the co-delivery of chemical drugs and nucleic acids for the treatment of RA cooperating with NIR-induced phototherapy.

Keywords:  RNA interference; hypoxia-activated chemotherapy; nano-delivery system; phototherapy; rheumatoid arthritis

DOI:  https://doi.org/10.2147/IJN.S382252
Biotechnol J. 2022 Dec 21. e2200415

Analysis of polymeric nanoparticle properties for siRNA/DNA delivery in a tumor xenograft tissue slice air-liquid interface model.

Michael Karimov, Marlene Scherer, Heike Franke, Alexander Ewe, Achim Aigner.

   BACKGROUND: Classical two-dimensional (2D) cell culture as a drug or nanoparticle test system only poorly recapitulates in vivo conditions. Animal studies are costly, ethically controversial, and preclude large-scale testing.
METHODS AND RESULTS: We established a three-dimensional (3D) tissue slice air-liquid interface (ALI) culture model for nanoparticle testing. We developed an optimized procedure for the reproducible generation of large sets of tissue slices from tumor xenografts that retain their tissue architecture. When used for the analysis of nanoparticles based on chemically modified polyethylenimines (PEIs) to deliver siRNA or DNA, differences in transfection efficacy and cytotoxicity between nanoparticles were observed more clearly than in 2D cell culture. While nanoparticle efficacies between cell culture and the tissue slice model overall correlated, the tissue slice model also identified particularly suitable candidates whose efficacy was underestimated in 2D cell culture and had already been shown in previous in vivo studies.
CONCLUSION: The ex vivo 3D tissue slice ALI culture model is a powerful system that allows the effective evaluation of biological nanoparticle efficacy and biocompatibility in an intact tissue environment. It is comparably inexpensive, time-saving, and follows the 3R principle, while allowing the identification of critical nanoparticle properties and optimal candidates for in vivo applications. This article is protected by copyright. All rights reserved.

Keywords:  PEI-based nanoparticles; Tissue slices; air-liquid interface culture; gene knockdown; gene transfection; polyethylenimine

DOI:  https://doi.org/10.1002/biot.202200415
Adv Healthc Mater. 2022 Dec 19. e2202528

Delivering mRNA to Secondary Lymphoid Tissues by Phosphatidylserine-loaded Lipid Nanoparticles.

Masaki Gomi, Yu Sakurai, Minami Sato, Hiroki Tanaka, Yumi Miyatake, Koichi Fujiwara, Mizuki Watanabe, Satoshi Shuto, Yuta Nakai, Kota Tange, Hiroto Hatakeyama, Hidetaka Akita.

  Lipid nanoparticles (LNPs) are one of the most successful technologies in mRNA delivery. While the liver has been the most frequent target for LNP delivery of mRNA, technologies for delivering mRNA molecules to extrahepatic tissues are also important. Herein, we report on the development of a LNP that targets secondary lymphoid tissues. We designed new types of alcohol-soluble phosphatidylserine (PS) derivatives as materials which target immune cells, and then incorporated them into LNPs using a microfluidic technique with a high degree of scalability and reproducibility. The resulting LNP that contained the synthesized PS delivered mRNA to the spleen much more efficiently compared to a control LNP. A sub-organ analysis revealed that the PS-loaded LNP was extensively taken up by tissue-resident macrophages in the red pulp and the marginal zone of the spleen. Thus, the PS-loaded LNP reported in this study would be a promising strategy for clinical applications that involve delivering mRNA to the spleen. This article is protected by copyright. All rights reserved.

Keywords:  Lipid nanoparticles; lymph node; mRNA delivery; phosphatidylserine; spleen

DOI:  https://doi.org/10.1002/adhm.202202528
Sci Adv. 2022 Dec 21. 8(51): eabq3699

Nanoparticle delivery of CD40 siRNA suppresses alloimmune responses by inhibiting activation and differentiation of DCs and macrophages.

Jialiang Wang, Kuirong Mao, Xiuxiu Cong, Huizhu Tan, Chenxi Wu, Zheng Hu, Yong-Guang Yang, Tianmeng Sun.

CD40 is an important costimulatory molecule expressed on antigen-presenting cells (APCs) and plays a critical role for APC activation, offering a promising therapeutic target for preventing allograft rejection. Here, we developed a biodegradable nanoparticle small interfering RNA delivery system (siCD40/NPs) to effectively deliver CD40 siRNA (siCD40) into hematopoietic stem cells (HSCs), myeloid progenitors, and mature dendritic cells (DCs) and macrophages. Injection of siCD40/NPs not only down-regulated CD40 expression in DCs and macrophages but also inhibited the differentiation of HSCs and/or myeloid progenitors into functional DCs and macrophages. Furthermore, siCD40/NPs treatment significantly prolonged allograft survival in mouse models of skin allotransplantation. In addition to reiteration of the role of CD40 in APC activation, our findings highlight a previously unappreciated role of CD40 in DC and macrophage differentiation from their progenitors. Furthermore, our results support the effectiveness of siCD40/NPs in suppressing alloimmune responses, providing a potential means of facilitating tolerance induction and preventing allotransplant rejection.

DOI: https://doi.org/10.1126/sciadv.abq3699
Biomaterials. 2022 Dec 13. pii: S0142-9612(22)00599-3. [Epub ahead of print]293 121959

Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins.

Jeanette M Metzger, Yuyuan Wang, Samuel S Neuman, Kathy J Snow, Stephen A Murray, Cathleen M Lutz, Viktoriya Bondarenko, Jesi Felton, Kirstan Gimse, Ruosen Xie, Dongdong Li, Yi Zhao, Matthew T Flowers, Heather A Simmons, Subhojit Roy, Krishanu Saha, Jon E Levine, Marina E Emborg, Shaoqin Gong.

  Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. In vivo delivery systems for CRISPR-Cas proteins and single guide RNA (sgRNA) include both viral vectors and non-viral strategies, each presenting different advantages and disadvantages for clinical application. We developed non-viral and biodegradable PEGylated nanocapsules (NCs) that deliver preassembled Cas9-sgRNA ribonucleoproteins (RNPs). Here, we show that the RNP NCs led to robust genome editing in neurons following intracerebral injection into the healthy mouse striatum. Genome editing was predominantly observed in medium spiny neurons (>80%), with occasional editing in cholinergic, calretinin, and parvalbumin interneurons. Glial activation was minimal and was localized along the needle tract. Our results demonstrate that the RNP NCs are capable of safe and efficient neuronal genome editing in vivo.

Keywords:  Brain; CRISPR; Genome editing; Nanocapsule; Nanoparticle; Neuron

DOI:  https://doi.org/10.1016/j.biomaterials.2022.121959
Sci Adv. 2022 Dec 23. 8(51): eabq3500

Manganese-coordinated mRNA vaccines with enhanced mRNA expression and immunogenicity induce robust immune responses against SARS-CoV-2 variants.

Na Fan, Kepan Chen, Rong Zhu, Zhongwei Zhang, Hai Huang, Shugang Qin, Qian Zheng, Zhongshan He, Xi He, Wen Xiao, Yupei Zhang, Yongjun Gu, Changchun Zhao, Yongmei Liu, Xin Jiang, Shuaicheng Li, Yuquan Wei, Xiangrong Song.

It is urgent to develop more effective mRNA vaccines against the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants owing to the immune escape. Here, we constructed a novel mRNA delivery system [IC8/Mn lipid nanoparticles (IC8/Mn LNPs)]with high immunogenicity, via introducing a stimulator of interferon genes (STING) agonist [manganese (Mn)] based on a newly synthesized ionizable lipid (IC8). It was found that Mn can not only promote maturation of antigen-presenting cells via activating STING pathway but also improve mRNA expression by facilitating lysosomal escape for the first time. Subsequently, IC8/Mn LNPs loaded with mRNA encoding the Spike protein of SARS-CoV-2 Delta or Omicron variant (IC8/Mn@D or IC8/Mn@O) were prepared. Both mRNA vaccines induced substantial specific immunoglobulin G responses against Delta or Omicron. IC8/Mn@D displayed strong pseudovirus neutralization ability, T helper 1-biased immune responses, and good safety. It can be concluded that IC8/Mn LNPs have great potential for developing Mn-coordinated mRNA vaccines with robust immunogenicity and good safety.

DOI: https://doi.org/10.1126/sciadv.abq3500