bims-novged 2023-04-23 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023–04–23
ten papers selected by
the Merkel lab, Ludwig-Maximilians University

Electrostatic Attractive Self-Delivery of siRNA and Light-Induced Self-Escape for Synergistic Gene Therapy.
Ratiometric, pH-Sensitive Probe for Monitoring siRNA Delivery.
Activatable NIR-II Photothermal Lipid Nanoparticles for Improved Messenger RNA Delivery.
Successful batch and continuous lyophilization of mRNA LNP formulations depend on cryoprotectants and ionizable lipids.
CES1-Triggered Liver-Specific Cargo Release of CRISPR/Cas9 Elements by Cationic Triadic Copolymeric Nanoparticles Targeting Gene Editing of PCSK9 for Hyperlipidemia Amelioration.
Cationic lipid-assisted nanoparticles for simultaneous delivery of CD47 siRNA and R848 to promote antitumor immune responses.
Tumor immunosuppression relief via acidity modulation combined PD-L1 siRNA for enhanced immunotherapy.
Immunogenicity and protective efficacy of SARS-CoV-2 mRNA vaccine encoding secreted non-stabilized spike in female mice.
Biomimetic Nanoparticle-Mediated Target Delivery of Hypoxia-Responsive Plasmid of Angiotensin-Converting Enzyme 2 to Reverse Hypoxic Pulmonary Hypertension.
A Fluorophore-Labeled Lysine Dendrimer with an Oxo-Anion-Binding Motif for Tracking Gene Transfection.

Adv Mater. 2023 Apr 21. e2301409

Electrostatic Attractive Self-Delivery of siRNA and Light-Induced Self-Escape for Synergistic Gene Therapy.

Yuxin Yang, Haijun Ning, Tianping Xia, Jianjun Du, Wen Sun, Jiangli Fan, Xiaojun Peng.

  Small interfering RNA (siRNA) holds immense promise for suppressing gene expression and treating various life-threatening diseases, including cancer. However, efficient delivery and lysosomal escape remain critical challenges that hinder the therapeutic effectiveness of siRNA. Herein, cationic photosensitizer (NB-Br) was grafted onto polo-like kinase 1 (PLK1) siRNA to form an amphiphilic siRNA-photosensitizer conjugate (siPLK1-NB), which could self-assemble into nanoparticles (siPLK1-NB NPs) via electrostatic attraction. Notably, siPLK1-NB NPs exhibited rapid and efficient cell endocytosis, as well as outstanding tumor-targeting property in multiple tumor-bearing mice models. When siPLK1-NB NPs were located inside tumor cell lysosomes, the generated reactive oxygen species (ROS) after photoactivation could disrupt the lysosome membrane structure and facilitate siRNA escape from lysosomes. Under light irradiation, siPLK1-NB NPs could downregulate PLK1 expression and induce photodynamic killing, effectively inhibiting tumor cell growth both in vitro and in vivo. Consequently, this study provides a novel design strategy for carrier-free siRNA delivery systems. To the best of our knowledge, this is the first report of a carrier-free siRNA delivery system based on electrostatic attraction. This article is protected by copyright. All rights reserved.

Keywords:  gene therapy; lysosome escape; photodynamic therapy; self-delivery; siRNA

DOI:  https://doi.org/10.1002/adma.202301409
J Am Chem Soc. 2023 Apr 19.

Ratiometric, pH-Sensitive Probe for Monitoring siRNA Delivery.

Madison R Herling, Ivan J Dmochowski.

Many RNA delivery strategies require efficient endosomal uptake and release. To monitor this process, we developed a 2'-OMe RNA-based ratiometric pH probe with a pH-invariant 3'-Cy5 and 5'-FAM whose pH sensitivity is enhanced by proximal guanines. The probe, in duplex with a DNA complement, exhibits a 48.9-fold FAM fluorescence enhancement going from pH 4.5 to pH 8.0 and reports on both endosomal entrapment and release when delivered to HeLa cells. In complex with an antisense RNA complement, the probe constitutes an siRNA mimic capable of protein knockdown in HEK293T cells. This illustrates a general approach for measuring the localization and pH microenvironment of any oligonucleotide.

DOI: https://doi.org/10.1021/jacs.3c01032
Angew Chem Int Ed Engl. 2023 Apr 19. e202302676

Activatable NIR-II Photothermal Lipid Nanoparticles for Improved Messenger RNA Delivery.

Benhao Li, Mengyao Zhao, Weiping Lai, Xuanbo Zhang, Bowei Yang, Xiaoyuan Chen, Qianqian Ni.

  Endosomal escape remains a central issue limiting the high protein expression of mRNA therapeutics. Here, we present second near-infrared (NIR-II) lipid nanoparticles (LNPs) containing pH activatable NIR-II dye conjugated lipid (Cy-lipid) for potentiating mRNA delivery efficiencyvia stimulus-responsive photothermal-promoted endosomal escape delivery (SPEED) strategy. In acidic endosomal microenvironment, Cy-lipid is protonated and turns on NIR-II absorption for light-to-heat transduction mediated by 1064 nm laser irradiation. Then, the heat-promoted LNPs morphology change triggers rapid escape of NIR-II LNPs from the endosome, allowing about 3-fold enhancement of enhanced green fluorescent protein (eGFP) encoding mRNA translation capacity compared to the NIR-II light free group. And the bioluminescence intensity induced by delivered luciferase encoding mRNA in mouse liver region shows positive correlation with incremental radiation dose, indicating the validity of the SPEED strategy.

Keywords:  endosomal escape; lipid nanoparticle; photothermal; second near-infrared window

DOI:  https://doi.org/10.1002/anie.202302676
Biomater Sci. 2023 Apr 19.

Successful batch and continuous lyophilization of mRNA LNP formulations depend on cryoprotectants and ionizable lipids.

Alexander Lamoot, Joris Lammens, Emily De Lombaerde, Zifu Zhong, Mark Gontsarik, Yong Chen, Thomas R M De Beer, Bruno G De Geest.

The limited thermostability and need for ultracold storage conditions are the major drawbacks of the currently used nucleoside-modified lipid nanoparticle (LNP)-formulated messenger RNA (mRNA) vaccines, which hamper the distribution of these vaccines in low-resource regions. The LNP core contains, besides mRNA and lipids, a large fraction of water. Therefore, encapsulated mRNA, or at least a part of it, is subjected to hydrolysis mechanisms similar to unformulated mRNA in an aqueous solution. It is likely that the hydrolysis of mRNA and colloidal destabilization are critical factors that decrease the biological activity of mRNA LNPs upon storage under ambient conditions. Hence, lyophilization as a drying technique is a logical and appealing method to improve the thermostability of these vaccines. In this study, we demonstrate that mRNA LNP formulations comprising a reduction-sensitive ionizable lipid can be successfully lyophilized, in the presence of 20% w/v sucrose, both by conventional batch freeze-drying and by an innovative continuous spin lyophilization process. While the chemical structure of the ionizable lipid did not affect the colloidal stability of the LNP after lyophilization and redispersion in an aqueous medium, we found that the ability of LNPs to retain the mRNA payload stably encapsulated, and mediate in vivo and in vitro mRNA translation into protein, post lyophilization strongly depended on the ionizable lipid in the LNP formulation.

DOI: https://doi.org/10.1039/d2bm02031a
Adv Sci (Weinh). 2023 Apr 21. e2300502

CES1-Triggered Liver-Specific Cargo Release of CRISPR/Cas9 Elements by Cationic Triadic Copolymeric Nanoparticles Targeting Gene Editing of PCSK9 for Hyperlipidemia Amelioration.

Yunfei Zhao, Yun Li, Fan Wang, Xuelan Gan, Tianye Zheng, Mengyue Chen, Li Wei, Jun Chen, Chao Yu.

  The broad application of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing tools is hindered by challenges in the efficient delivery of its two components into specific cells and intracytoplasmic release. Herein, a novel copolymer for delivery of Cas9-mRNA/ single-guide RNA (Cas9-mRNA/sgRNA) in vitro and vivo, using carboxylesterase-responsive cationic triadic copolymeric nanoparticles targeted proprotein convertase subtilisin/kexin type 9 (PCSK9) for hyperlipidemia amelioration is reported. A dimethyl biguanide derivative is designed and synthesized to form cationic block, and copolymerization onto prepolymer with propyl methacrylate, to fabricate a triadic copolymer mPEG-b-P(Met/n-PMA). The copolymer can self-assemble with Cas9-mRNA/sgRNA, indicating the excellent potential of nanoparticles to form a delivery carrier. This vehicle can efficiently release RNA in response to the hepatocytes carboxylesterase for genome editing. It was demonstrated that the mPEG-b-P(Met/n-PMA)/Cas9 mRNA/sgRNA nanoparticles effectively accumulated in hepatocytes, lead to the inhibition of PCSK9, and lowered the levels of Low-density lipoprotein cholesterol and total cholesterol in mouse serum down 20% of nontreatment. Interestingly, the nanoparticles even enable multiple functions in the regulation of blood glucose and weight. This study establishes a novel method to achieve complex CRISPR components stable loading, safe delivery, and fixed-point release, which expand the application of CRISPR delivery systems.

Keywords:  CES active destabilized strategy; CRISPR/Cas9; PCSK9; hyperlipidemia amelioration; liver fixed-point release; mRNA delivery

DOI:  https://doi.org/10.1002/advs.202300502
Front Pharmacol. 2023 ;14 1142374

Cationic lipid-assisted nanoparticles for simultaneous delivery of CD47 siRNA and R848 to promote antitumor immune responses.

Simin Li, Yichang Chen, Ruolin Ma, Ye Du, Bing Han.

  Introduction: Triple-negative breast cancer (TNBC) usually has a poor prognosis. Although the immunotherapy of TNBC has achieved great progress, only a few patients can benefit from the current treatment. CD47 is widely expressed on the surface of TNBC cells and may become an immune checkpoint for TNBC treatment. Nevertheless, increasingly more attention has been paid to systemic side effects since the ubiquitous expression of CD47 on normal cells. The toll-like receptor (TLR) agonist resiquimod (R848) can activate dendritic cells (DCs) and promote the maturation of immune cells in the tumor microenvironment, which further enhances the tumor inhibition ability of the immune system and synergizes with CD47 small interfering RNA (siRNA) for TNBC therapy. However, ideal delivery platforms such as nanocarriers are still needed because its weakness of hydrophobicity. Methods: In order to improve efficacy and reduce toxicity, R848 and siCD47 were entrapped in amphiphilic PEG-PLGA nanoparticles by double emulsification and stable nanoparticles NP/R848/siCD47 were generated to investigate their anti-tumor effects in a TNBC tumor-bearing mouse model. Results: Here, we show that PEG-PLGA nanoparticles are effective nanocarriers that can safely and effectively deliver siCD47 and R848 to tumor tissue, as demonstrated by retarded tumor growth. Mechanistically, downregulation of CD47 expression and activation of DCs took part in promoting the immune response of cytotoxic T cells (CTLs). Meanwhile, a decrease of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) indicated the modulating of the tumor immune microenvironment. Discussion: To our best knowledge, our study pioneered co-delivery system for hydrophilic siCD47 and hydrophobic R848. It can maximize break tumor immune escape caused by CD47 and simultaneously enhance antigen presentation by activating DCs for effector T cell killing while regulating the tumor microenvironment as expected. Not only does it conform to the reports of previous basic research, but also it can break the bottleneck of their clinical application hopefully. Collectively, our findings could lay the foundation for future therapeutic strategies of TNBC.

Keywords:  PEG-PLGA nanoparticles; R848; TNBC; siCD47; tumor immunotherapy

DOI:  https://doi.org/10.3389/fphar.2023.1142374
Biomater Adv. 2023 Apr 11. pii: S2772-9508(23)00148-6. [Epub ahead of print]150 213425

Tumor immunosuppression relief via acidity modulation combined PD-L1 siRNA for enhanced immunotherapy.

Yan Tang, Qingcheng Chang, Gang Chen, Xiaomei Zhao, Gui Huang, Tong Wang, Changhao Jia, Linghong Lu, Taiwei Jin, Shudi Yang, Li Cao, Xuenong Zhang.

  The efficacy of immune checkpoint therapy is limited by the immunosuppressive tumor microenvironment (TME), and lactate, the most universal component of TME, has been rediscovered that plays important roles in the regulation of metabolic pathways, angiogenesis, and immunosuppression. Here, a therapeutic strategy of acidity modulation combined with programmed death ligand-1 (PD-L1) siRNA (siPD-L1) is proposed to synergistically enhance tumor immunotherapy. The lactate oxidase (LOx) is encapsulated into the hollow Prussian blue (HPB) nanoparticles (NPs) prepared by hydrochloric acid etching followed by the modification with polyethyleneimine (PEI) and polyethylene glycol (PEG) via sulfur bonds (HPB-S-PP@LOx), siPD-L1 is loaded via electrostatic adsorption to obtain HPB-S-PP@LOx/siPD-L1. The obtained co-delivery NPs can accumulate in tumor tissue with stable systemic circulation, and simultaneous release of LOx and siPD-L1 in intracellular high glutathione (GSH) environment after uptake by tumor cells without being destroyed by lysosome. Moreover, LOx can catalyze the decomposition of lactate in the hypoxic tumor tissue with the aid of oxygen release by the HPB-S-PP nano-vector. The results show that the acidic TME regulation via lactate consumption can improve the immunosuppressive TME, including revitalizing the exhausted CD8+ T cells and decreasing the proportion of immunosuppressive Tregs, and synergistically elevating the therapeutic effect of PD1/PD-L1 blockade therapy via siPD-L1. This work provides a novel insight for tumor immunotherapy and explores a promising therapy for triple-negative breast cancer.

Keywords:  Acidity modulation; Co-delivery system; Tumor immunotherapy; siPD-L1

DOI:  https://doi.org/10.1016/j.bioadv.2023.213425
Nat Commun. 2023 Apr 21. 14(1): 2309

Immunogenicity and protective efficacy of SARS-CoV-2 mRNA vaccine encoding secreted non-stabilized spike in female mice.

Eakachai Prompetchara, Chutitorn Ketloy, Mohamad-Gabriel Alameh, Kittipan Tharakhet, Papatsara Kaewpang, Nongnaphat Yostrerat, Patrawadee Pitakpolrat, Supranee Buranapraditkun, Suwimon Manopwisedjaroen, Arunee Thitithanyanont, Anan Jongkaewwattana, Taweewan Hunsawong, Rawiwan Im-Erbsin, Matthew Reed, Wassana Wijagkanalan, Kanitha Patarakul, Teerasit Techawiwattanaboon, Tanapat Palaga, Kieu Lam, James Heyes, Drew Weissman, Kiat Ruxrungtham.

Establishment of an mRNA vaccine platform in low- and middle-income countries (LMICs) is important to enhance vaccine accessibility and ensure future pandemic preparedness. Here, we describe the preclinical studies of "ChulaCov19", a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP). In female BALB/c mice, ChulaCov19 at 0.2, 1, 10, and 30 μg elicits robust neutralizing antibody (NAb) and T cell responses in a dose-dependent relationship. The geometric mean titers (GMTs) of NAb against wild-type (WT, Wuhan-Hu1) virus are 1,280, 11,762, 54,047, and 62,084, respectively. Higher doses induce better cross-NAb against Delta (B.1.617.2) and Omicron (BA.1 and BA.4/5) variants. This elicited immunogenicity is significantly higher than those induced by homologous CoronaVac or AZD1222 vaccination. In a heterologous prime-boost study, ChulaCov19 booster dose generates a 7-fold increase of NAb against Wuhan-Hu1 WT virus and also significantly increases NAb response against Omicron (BA.1 and BA.4/5) when compared to homologous CoronaVac or AZD1222 vaccination. Challenge studies show that ChulaCov19 protects human-ACE-2-expressing female mice from COVID-19 symptoms, prevents viremia and significantly reduces tissue viral load. Moreover, anamnestic NAb response is undetectable in challenge animals. ChulaCov19 is therefore a promising mRNA vaccine candidate either as a primary or boost vaccination and has entered clinical development.

DOI: https://doi.org/10.1038/s41467-023-37795-0
ACS Nano. 2023 Apr 18.

Biomimetic Nanoparticle-Mediated Target Delivery of Hypoxia-Responsive Plasmid of Angiotensin-Converting Enzyme 2 to Reverse Hypoxic Pulmonary Hypertension.

Rui Yuan, Manling Liu, Ying Cheng, Fang Yan, Xiaoquan Zhu, Siyuan Zhou, Mingqing Dong.

  Hypoxic pulmonary hypertension (HPH) is characterized by pulmonary vascular sustained constriction and progressive remodeling, which are initiated by hypoxia then with hypoxia-induced additive factors including pulmonary vascular endothelium injury, intrapulmonary angiotension system imbalance, and inflammation. Now HPH is still an intractable disease lacking effective treatments. Gene therapy has a massive potential for HPH but is hindered by a lack of efficient targeted delivery and hypoxia-responsive regulation systems for transgenes. Herein, we constructed the hypoxia-responsive plasmid of angiotensin-converting enzyme 2 (ACE2) with endothelial-specific promoter Tie2 and a hypoxia response element and next prepared its biomimetic nanoparticle delivery system, named ACE2-CS-PRT@PM, by encapsulating the plasmid of ACE2 with protamine and chondroitin sulfate as the core then coated it with a platelet membrane as a shell for targeting the injured pulmonary vascular endothelium. ACE2-CS-PRT@PM has a 194.3 nm diameter with a platelet membrane-coating core-shell structure and a negatively charged surface, and it exhibits higher delivery efficiency targeting to pulmonary vascular endothelium and hypoxia-responsive overexpression of ACE2 in endothelial cells in a hypoxia environment. In vitro, ACE2-CS-PRT@PM significantly inhibited the hypoxia-induced proliferation of pulmonary smooth muscle cells. In vivo, ACE2-CS-PRT@PM potently ameliorated the hemodynamic dysfunction and morphological abnormality and largely reversed HPH via inhibiting the hypoxic proliferation of pulmonary artery smooth muscle cells, reducing pulmonary vascular remodeling, restoring balance to the intrapulmonary angiotension system, and improving the inflammatory microenvironment without any detectable toxicity. Therefore, ACE2-CS-PRT@PM is promising for the targeted gene therapy of HPH.

Keywords:  angiotensin-converting enzyme 2; biomimetic nanoparticle; hypoxia-responsive plasmid; hypoxic pulmonary hypertension; inflammatory microenvironment; platelet membrane coating

DOI:  https://doi.org/10.1021/acsnano.2c12190
Chembiochem. 2023 Apr 18. e202300296

A Fluorophore-Labeled Lysine Dendrimer with an Oxo-Anion-Binding Motif for Tracking Gene Transfection.

Nazli Aldemir, Cecilia Vallet, Shirley Knauer, Carsten Schmuck, Christoph Hirschhäuser.

  A transfection vector based on a peptide dendrimer (1) was developed and its ability for DNA binding and transport investigated. By attaching a fluorophore to the vector system (1*), several steps in the transfection process could be monitored directly. As DLS and AFM studies showed, the labeled vector1*condensed DNA into tightly packed aggregates able to enter eukaryotic cells. Co-localization experiments revealed that the ligand/plasmid complex is taken upviathe endosomal pathway followed by an endosomal escape or lysosomal degradation. Afterwards, the plasmid DNA seems to enter the nucleus due to a breakdown of the nuclear envelope during mitosis, as only cells that have recently undergone mitosis showed H2B-GFP expression.

Keywords:  gene transfection, nonviral vector, tracking, DNA, binding ligand

DOI:  https://doi.org/10.1002/cbic.202300296