bims-novged 2021-06-20 papers

on Non-viral vectors for gene delivery

Issue of 2021–06–20
twelve papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

All-In-One Dendrimer-Based Lipid Nanoparticles Enable Precise HDR-Mediated Gene Editing In Vivo.
Conformation-sensitive targeting of lipid nanoparticles for RNA therapeutics.
Identification of a potent ionizable lipid for efficient macrophage transfection and systemic anti-interleukin-1β siRNA delivery against acute liver failure.
Periodic Mesoporous Ionosilica Nanoparticles for Green Light Photodynamic Therapy and Photochemical Internalization of siRNA.
SiRNA in MSC-derived exosomes silences CTGF gene for locomotor recovery in spinal cord injury rats.
Intracellular Delivery of mRNA in Adherent and Suspension Cells by Vapor Nanobubble Photoporation.
Highly Efficient Transfection of Human Primary T Lymphocytes Using Droplet-Enabled Mechanoporation.
Exosomes as Natural Delivery Carriers for Programmable Therapeutic Nucleic Acid Nanoparticles (NANPs).
Improved cardiac-specific delivery of RAGE siRNA within small extracellular vesicles engineered to express intense cardiac targeting peptide attenuates myocarditis.
Fluorescence lifetime imaging microscopy (FLIM) of intracellular transport by means of doubly labelled siRNA architectures.
mRNA Delivery by a pH-Responsive DNA Nano-Hydrogel.
In Situ Tumor Vaccination with Nanoparticle Co-Delivering CpG and STAT3 siRNA to Effectively Induce Whole-Body Antitumor Immune Response.

Adv Mater. 2021 Jun 17. e2006619

All-In-One Dendrimer-Based Lipid Nanoparticles Enable Precise HDR-Mediated Gene Editing In Vivo.

Lukas Farbiak, Qiang Cheng, Tuo Wei, Ester Álvarez-Benedicto, Lindsay T Johnson, Sang Lee, Daniel J Siegwart.

  Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein gene editing is poised to transform the treatment of genetic diseases. However, limited progress has been made toward precise editing of DNA via homology-directed repair (HDR) that requires careful orchestration of complex steps. Herein, dendrimer-based lipid nanoparticles (dLNPs) are engineered to co-encapsulate and deliver multiple components for in vivo HDR correction. BFP/GFP switchable HEK293 cells with a single Y66H amino acid mutation are employed to assess HDR-mediated gene editing following simultaneous, one-pot delivery of Cas9 mRNA, single-guide RNA, and donor DNA. Molar ratios of individual LNP components and weight ratios of the three nucleic acids are systematically optimized to increase HDR efficiency. Using flow cytometry, fluorescence imaging, and DNA sequencing to quantify editing, optimized 4A3-SC8 dLNPs edit >91% of all cells with 56% HDR efficiency in vitro and >20% HDR efficiency in xenograft tumors in vivo. Due to the all-in-one simplicity and high efficacy, the developed dLNPs offer a promising route toward the gene correction of disease-causing mutations.

Keywords:  CRISPR/Cas; gene editing; mRNA delivery; nanoparticles; nucleic acid delivery

DOI:  https://doi.org/10.1002/adma.202006619
Nat Nanotechnol. 2021 Jun 17.

Conformation-sensitive targeting of lipid nanoparticles for RNA therapeutics.

Niels Dammes, Meir Goldsmith, Srinivas Ramishetti, Jason L J Dearling, Nuphar Veiga, Alan B Packard, Dan Peer.

The successful in vivo implementation of gene expression modulation strategies relies on effective, non-immunogenic delivery vehicles. Lipid nanoparticles are one of the most advanced non-viral clinically approved nucleic-acid delivery systems. Yet lipid nanoparticles accumulate naturally in liver cells upon intravenous administration, and hence, there is an urgent need to enhance uptake by other cell types. Here we use a conformation-sensitive targeting strategy to achieve in vivo gene silencing in a selective subset of leukocytes and show potential therapeutic applications in a murine model of colitis. In particular, by targeting the high-affinity conformation of α4β7 integrin, which is a hallmark of inflammatory gut-homing leukocytes, we silenced interferon-γ in the gut, resulting in an improved therapeutic outcome in experimental colitis. The lipid nanoparticles did not induce adverse immune activation or liver toxicity. These results suggest that our lipid nanoparticle targeting strategy might be applied for selective delivery of payloads to other conformation-sensitive targets.

DOI: https://doi.org/10.1038/s41565-021-00928-x
J Mater Chem B. 2021 Jun 16.

Identification of a potent ionizable lipid for efficient macrophage transfection and systemic anti-interleukin-1β siRNA delivery against acute liver failure.

Feng Ding, Hongqian Zhang, Qiang Li, Chuanxu Yang.

RNA interference (RNAi) therapy has great potential for treating inflammatory diseases. However, the development of potent carrier materials for delivering siRNA to macrophages is challenging. Herein, we design a set of ionizable lipid nanoparticles (LNPs) to screen and identify a potent carrier of siRNA for silencing an essential pro-inflammatory cytokine, interleukin-1β (IL-1β) in macrophages. The top performance LNP (114-LNP), containing ionizable lipid with spermine as an amine-head group, facilitated efficient siRNA internalization via multiple endocytosis pathways and achieved effective endosome escape in macrophages. The optimized LNP/siIL-1β achieved strong silencing of IL-1β in both activated Raw 264.7 cells and primary macrophages. Furthermore, systematic administration of 114-LNP/siIL-1β complexes could effectively inhibit IL-1β expression in an acute liver failure model and significantly attenuated hepatic inflammation and liver damage. These results suggest that the optimized ionizable lipid nanoparticle represents a promising platform for anti-inflammation therapies.

DOI: https://doi.org/10.1039/d1tb00736j
ACS Appl Mater Interfaces. 2021 Jun 17.

Periodic Mesoporous Ionosilica Nanoparticles for Green Light Photodynamic Therapy and Photochemical Internalization of siRNA.

Braham Mezghrani, Lamiaa M A Ali, Sébastien Richeter, Jean-Olivier Durand, Peter Hesemann, Nadir Bettache.

  We report periodic mesoporous ionosilica nanoparticles (PMINPs) as versatile nano-objects for imaging, photodynamic therapy (PDT), and efficient adsorption and delivery of small interfering RNA (siRNA) into breast cancer cells. In order to endow these nanoparticles with PDT and siRNA photochemical internalization (PCI) properties, a porphyrin derivative was integrated into the ionosilica framework. For this purpose, we synthesized PMINPs via hydrolysis-cocondensation procedures from oligosilylated ammonium and porphyrin precursors. The formation of these nano-objects was proved by transmission electron microscopy. The formed nanoparticles were then thoroughly characterized via solid-state NMR, nitrogen sorption, dynamic light scattering, and UV-vis and fluorescence spectroscopies. Our results indicate the formation of highly porous nanorods with a length of 108 ± 9 nm and a width of 54 ± 4 nm. A significant PDT effect of type I mechanism (95 ± 2.8% of cell death) was observed upon green light irradiation in nanoparticle-treated breast cancer cells, while the blue light irradiation caused a significant phototoxic effect in non-treated cells. Furthermore, PMINPs formed stable complexes with siRNA (up to 24 h), which were efficiently internalized into the cells after 4 h of incubation mostly with the energy-dependent endocytosis process. The PCI effect was obvious with green light irradiation and successfully led to 83 ± 1.1% silencing of the luciferase gene in luciferase-expressing breast cancer cells, while no gene silencing effect was observed with blue light irradiation. The present work highlights the high potential of porphyrin-doped PMINPs as multifunctional nanocarriers for nucleic acids, such as siRNA, with a triple ability to perform imaging, PDT, and PCI.

Keywords:  gene silencing; periodic mesoporous ionosilica nanoparticles; photochemical internalization; photodynamic therapy; siRNA

DOI:  https://doi.org/10.1021/acsami.1c05848
Stem Cell Res Ther. 2021 Jun 10. 12(1): 334

SiRNA in MSC-derived exosomes silences CTGF gene for locomotor recovery in spinal cord injury rats.

Wei Huang, Mingjia Qu, Lu Li, Tao Liu, Miaoman Lin, Xiaobing Yu.

   BACKGROUND: How to obtain a small interfering RNA (siRNA) vector has become a moot point in recent years. Exosomes (Exo) show advantages of long survival time in vivo, high transmission efficiency, and easy penetration across the blood-spinal cord barrier, renowned as excellent carriers of bioactive substances.
METHODS: We applied mesenchymal stem cell (MSC)-derived exosomes as the delivery of synthesized siRNA, which were extracted from rat bone marrow. We constructed exosomes-siRNA (Exo-siRNA) that could specifically silence CTGF gene in the injury sites by electroporation. During the administration, we injected Exo-siRNA into the tail vein of SCI rats, RESULTS: In vivo and in vitro experiments showed that Exo-siRNA not only effectively inhibited the expressions of CTGF gene, but quenched inflammation, and thwarted neuronal apoptosis and reactive astrocytes and glial scar formation. Besides, it significantly upregulated several neurotrophic factors and anti-inflammatory factors, acting as a facilitator of locomotor recovery of rats with spinal cord injury (SCI).
CONCLUSIONS: In conclusion, this study has combined the thoroughness of gene therapy and the excellent drug-loading characteristics of Exo for the precise treatment of SCI, which will shed new light on the drug-loading field of Exo.

Keywords:  Exosomes: Mesenchymal stem cells; RNA interference; Small interfering RNA; Spinal cord injury

DOI:  https://doi.org/10.1186/s13287-021-02401-x
Nanomicro Lett. 2020 Sep 27. 12(1): 185

Intracellular Delivery of mRNA in Adherent and Suspension Cells by Vapor Nanobubble Photoporation.

Laurens Raes, Stephan Stremersch, Juan C Fraire, Toon Brans, Glenn Goetgeluk, Stijn De Munter, Lien Van Hoecke, Rein Verbeke, Jelter Van Hoeck, Ranhua Xiong, Xavier Saelens, Bart Vandekerckhove, Stefaan De Smedt, Koen Raemdonck, Kevin Braeckmans.

  Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications, such as CAR T cell-based therapies. mRNA has recently gained increasing attention as a more safe and versatile alternative tool over viral- or DNA transposon-based approaches for the generation of adoptive T cells. However, limitations associated with existing nonviral mRNA delivery approaches hamper progress on genetic engineering of these hard-to-transfect immune cells. In this study, we demonstrate that gold nanoparticle-mediated vapor nanobubble (VNB) photoporation is a promising upcoming physical transfection method capable of delivering mRNA in both adherent and suspension cells. Initial transfection experiments on HeLa cells showed the importance of transfection buffer and cargo concentration, while the technology was furthermore shown to be effective for mRNA delivery in Jurkat T cells with transfection efficiencies up to 45%. Importantly, compared to electroporation, which is the reference technology for nonviral transfection of T cells, a fivefold increase in the number of transfected viable Jurkat T cells was observed. Altogether, our results point toward the use of VNB photoporation as a more gentle and efficient technology for intracellular mRNA delivery in adherent and suspension cells, with promising potential for the future engineering of cells in therapeutic and fundamental research applications.

Keywords:  Gold nanoparticles; Optoporation; Photoporation; Transfection; Vapor nanobubbles; mRNA

DOI:  https://doi.org/10.1007/s40820-020-00523-0
ACS Nano. 2021 Jun 18.

Highly Efficient Transfection of Human Primary T Lymphocytes Using Droplet-Enabled Mechanoporation.

Byeongju Joo, Jeongsoo Hur, Gi-Beom Kim, Seung Gyu Yun, Aram J Chung.

  Whole-cell-based therapy has been extensively used as an effective disease treatment approach, and it has rapidly changed the therapeutic paradigm. To fully accommodate this shift, advances in genome modification and cell reprogramming methodologies are critical. Traditionally, molecular tools such as viral and polymer nanocarriers and electroporation have been the norm for internalizing external biomolecules into cells for cellular engineering. However, these approaches are not fully satisfactory considering their cytotoxicity, high cost, low scalability, and/or inconsistent and ineffective delivery and transfection. To address these challenges, we present an approach that leverages droplet microfluidics with cell mechanoporation, bringing intracellular delivery to the next level. In our approach, cells and external cargos such as mRNAs and plasmid DNAs are coencapsulated into droplets, and as they pass through a series of narrow constrictions, the cell membrane is mechanically permeabilized where the cargos in the vicinity are internalized via convective solution exchange enhanced by recirculation flows developed in the droplets. Using this principle, we demonstrated a high level of functional macromolecule delivery into various immune cells, including human primary T cells. By utilizing droplets, the cargo consumption was drastically reduced, and near-zero clogging was realized. Furthermore, high scalability without sacrificing cell viability and superior delivery over state-of-the-art methods and benchtop techniques were demonstrated. Notably, the droplet-based intracellular delivery strategy presented here can be further applied to other mechanoporation microfluidic techniques, highlighting its potential for cellular engineering and cell-based therapies.

Keywords:  T cell engineering; cell therapy; droplet microfluidics; droplet squeezing; gene delivery; intracellular delivery; transfection

DOI:  https://doi.org/10.1021/acsnano.0c10473
Adv Drug Deliv Rev. 2021 Jun 15. pii: S0169-409X(21)00227-1. [Epub ahead of print] 113835

Exosomes as Natural Delivery Carriers for Programmable Therapeutic Nucleic Acid Nanoparticles (NANPs).

Weina Ke, Kirill A Afonin.

  With recent advances in nanotechnology and therapeutic nucleic acids (TNAs), various nucleic acid nanoparticles (NANPs) have demonstrated great promise in diagnostics and therapeutics. However, the full realization of NANPs' potential necessitates the development of a safe, efficient, biocompatible, stable, tissue-specific, and non-immunogenic delivery system. Exosomes, the smallest extracellular vesicles and an endogenous source of nanocarriers, offer these advantages while avoiding complications associated with manufactured agents. The lipid membranes of exosomes surround a hydrophilic core, allowing for the simultaneous incorporation of hydrophobic and hydrophilic drugs, nucleic acids, and proteins. Additional capabilities for post-isolation exosome surface modifications with imaging agents, targeting ligands, and covalent linkages also pave the way for their diverse biomedical applications. This review focuses on exosomes: their biogenesis, intracellular trafficking, transportation capacities, and applications with emphasis on the delivery of TNAs and programmable NANPs. We also highlight some of the current challenges and discuss opportunities related to the development of therapeutic exosome-based formulations and their clinical translation.

Keywords:  Nucleic acid nanoparticles (NANPs); Therapeutic Nucleic Acids (TNA); drug delivery; exosomes; extracellular vesicle; immunorecognition; nanotechnology

DOI:  https://doi.org/10.1016/j.addr.2021.113835
Mol Ther Nucleic Acids. 2021 Jun 04. 24 1024-1032

Improved cardiac-specific delivery of RAGE siRNA within small extracellular vesicles engineered to express intense cardiac targeting peptide attenuates myocarditis.

Hyoeun Kim, Dasom Mun, Ji-Young Kang, Seung-Hyun Lee, Nuri Yun, Boyoung Joung.

  Small extracellular vesicles (sEVs) are nanometer-sized membranous vesicles secreted by cells, with important roles in physiological and pathological processes. Recent research has established the application of sEVs as therapeutic vehicles in various conditions, including heart disease. However, the high risk of off-target effects is a major barrier for their introduction into the clinic. This study evaluated the use of modified sEVs expressing high levels of cardiac-targeting peptide (CTP) for therapeutic small interfering RNA (siRNA) delivery in myocarditis, an inflammatory disease of heart. sEVs were extracted from the cell culture medium of HEK293 cells stably expressing CTP-LAMP2b (referred to as C-sEVs). The cardiac targeting ability of C-sEVs with the highest CTP-LAMP2b expression was >2-fold greater than that of normal sEVs (N-sEVs). An siRNA targeting the receptor for advanced glycation end products (RAGE) (siRAGE) was selected as a therapeutic siRNA and loaded into C-sEVs. The efficiency of cardiac-specific siRNA delivery via C-sEVs was >2-fold higher than that via N-sEVs. Furthermore, siRAGE-loaded C-sEVs attenuated inflammation in both cell culture and an in vivo model of myocarditis. Taken together, C-sEVs may be a useful drug delivery vehicle for the treatment of heart disease.

Keywords:  cardiac-targeting peptide; heart-specific delivery; myocarditis; small extracellular vesicles; small interfering RNA

DOI:  https://doi.org/10.1016/j.omtn.2021.04.018
Chembiochem. 2021 Jun 14.

Fluorescence lifetime imaging microscopy (FLIM) of intracellular transport by means of doubly labelled siRNA architectures.

Larissa Doll, Jens Lackner, Franziska Rönicke, Hans-Achim Wagenknecht, Gerd Ulrich Nienhaus.

  For monitoring the intracellular pathway of small interfering RNA (siRNA), both strands were labelled at internal positions by two ATTO dyes as an interstrand Förster resonance energy transfer pair. siRNA double strands show red emission and a short donor lifetime as readout, whereas siRNA antisense single strands show green emission and a long donor lifetime. This readout signals if GFP silencing can be expected (green) or not (red). We attached both dyes to three structurally different alkyne anchors by postsynthetic modifications. There is only a slight preference for the ribofuranoside anchors with the dyes at their 2'-positions. For the first time, the delivery and fate of siRNA in live HeLa cells was tracked by FLIM, which revealed a clear relationship between intracellular transport using different transfection methods and knockdown of the GFP expression, which demonstrates the potential of our siRNA architectures as a tool for the future development of effective siRNA.

Keywords:  oligonucleotide * fluorescence * cycloaddition * transport * energy transfer

DOI:  https://doi.org/10.1002/cbic.202100150
Small. 2021 Jun 17. e2101224

mRNA Delivery by a pH-Responsive DNA Nano-Hydrogel.

Xin Fu, Tianshu Chen, Yuchen Song, Chang Feng, Huinan Chen, Qianqian Zhang, Guifang Chen, Xiaoli Zhu.

  The delivery of mRNA to manipulate protein expression has attracted widespread attention, since that mRNA overcomes the problem of infection and mutation risks in transgenes and can work as drugs for the treatment of diseases. Although there are currently some vehicles that deliver mRNA into cells, they have not yet reached a good balance in terms of expression efficiency and biocompatibility. Here, a DNA nano-hydrogel system for mRNA delivery is developed. The nano-hydrogel is all composed of DNA except the target mRNA, so it has superior biocompatibility compared with those chemical vehicles. In parallel, the nano-hydrogel can be compacted into a nanosphere under the crosslinking by well-designed "X"-shaped DNA scaffolds and DNA linkers, facilitating the delivery into cells through endocytosis. In addition, smart intracellular release of the mRNA is achieved by incorporating a pH-responsive i-motif structure into the nano-hydrogel. Thus, taking the efficient delivery and release together, mRNA can be translated into the corresponding protein with a high efficiency, which is comparable to that of the commercial liposome but with a much better biocompatibility. Due to the excellent biocompatibility and efficiency, this nano-hydrogel system is expected to become a competitive alternative for delivering functional mRNA in vivo.

Keywords:  Gaussia luciferase; delivery; green fluorescent proteins; mRNA; nano-hydrogels

DOI:  https://doi.org/10.1002/smll.202101224
Adv Mater. 2021 Jun 12. e2100628

In Situ Tumor Vaccination with Nanoparticle Co-Delivering CpG and STAT3 siRNA to Effectively Induce Whole-Body Antitumor Immune Response.

Worapol Ngamcherdtrakul, Moataz Reda, Molly A Nelson, Ruijie Wang, Husam Y Zaidan, Daniel S Bejan, Ngoc Ha Hoang, Ryan S Lane, Shiuh-Wen Luoh, Sancy A Leachman, Gordon B Mills, Joe W Gray, Amanda W Lund, Wassana Yantasee.

  The success of immunotherapy with immune checkpoint inhibitors (ICIs) in a subset of individuals has been very exciting. However, in many cancers, responses to current ICIs are modest and are seen only in a small subsets of patients. Herein, a widely applicable approach that increases the benefit of ICIs is reported. Intratumoral administration of augmenting immune response and inhibiting suppressive environment of tumors-AIRISE-02 nanotherapeutic that co-delivers CpG and STAT3 siRNA-results in not only regression of the injected tumor, but also tumors at distant sites in multiple tumor model systems. In particular, three doses of AIRISE-02 in combination with systemic ICIs completely cure both treated and untreated aggressive melanoma tumors in 63% of mice, while ICIs alone do not cure any mice. A long-term memory immune effect is also reported. AIRISE-02 is effective in breast and colon tumor models as well. Lastly, AIRISE-02 is well tolerated in mice and nonhuman primates. This approach combines multiple therapeutic agents into a single nanoconstruct to create whole-body immune responses across multiple cancer types. Being a local therapeutic, AIRISE-02 circumvents regulatory challenges of systemic nanoparticle delivery, facilitating rapid translation to the clinic. AIRISE-02 is under investigational new drug (IND)-enabling studies, and clinical trials will soon follow.

Keywords:  cancer immunotherapy; intratumoral therapy; melanoma; nanotechnology; translational research

DOI:  https://doi.org/10.1002/adma.202100628