bims-novged 2021-09-19 papers

on Non-viral vectors for gene delivery

Issue of 2021–09–19
nine papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

A novel β-glucan-oligonucleotide complex selectively delivers siRNA to APCs via Dectin-1.
Comparison of promoter, DNA vector, and cationic carrier for efficient transfection of hMSCs from multiple donors and tissue sources.
Dual-Modality Poly-l-histidine Nanoparticles to Deliver Peptide Nucleic Acids and Paclitaxel for In Vivo Cancer Therapy.
Radiosensitizing effect of gold nanoparticle loaded with small interfering RNA-SP1 on lung cancer: AuNPs-si-SP1 regulates GZMB for radiosensitivity.
Reduction-Sensitive Fluorinated-Pt(IV) Universal Transfection Nanoplatform Facilitating CT45-Targeted CRISPR/dCas9 Activation for Synergistic and Individualized Treatment of Ovarian Cancer.
Supramolecular self-assembled DNA nanosystem for synergistic chemical and gene regulations on cancer cells.
Engineered exosomes loaded with miR-449a selectively inhibit the growth of homologous non-small cell lung cancer.
Polyplex nanomicelle delivery of self-amplifying RNA vaccine.
Optimized two-step electroporation process to achieve efficient non-viral-mediated gene insertion into primary T cells.

J Control Release. 2021 Sep 13. pii: S0168-3659(21)00490-9. [Epub ahead of print]

A novel β-glucan-oligonucleotide complex selectively delivers siRNA to APCs via Dectin-1.

Atsushi Uno, Kenji Arima, Masako Shimazaki, Maki Ushida, Kanako Amano, Reiko Namikawa, Kazuo Sakurai.

  Delivering therapeutic nucleic acids to targeted cells and organs has been a challenge for decades. A novel technology to deliver oligonucleotide therapeutics to immune cells is here described. In this approach, a macromolecular complex of oligonucleotides and the β-1,3-glucan schizophyllan (SPG) is selectively delivered to cells expressing a lectin receptor, Dectin-1, via SPG-Dectin-1 interaction. Detailed investigation of Dectin-1-expressing cells revealed that Dectin-1 is expressed in all subsets of monocytes as well as dendritic cell (DC) populations, including conventional DCs (cDCs) and plasmacytoid DCs (pDCs), in humans. The expression patterns in mice and humans are comparable, except for the expression in pDCs. The results indicate that Dectin-1 is expressed on cells capable of professional antigen presentation, except for B cells. We chose CD40 as a target gene for small interfering RNA (siRNA) as CD40 expression in antigen-presenting cells (APCs), particularly in DCs, plays critical roles in regulating immune responses. Dose-dependent cellular uptake of siCD40-SPG complexes was confirmed in cells expressing Dectin-1. Gene silencing activity was confirmed in vitro by the reduction of CD40 mRNA and by the site-specific cleavage of CD40 mRNA as determined by the 5' RNA ligase-mediated rapid amplification of cDNA ends (5'RLM-RACE) technique. In vivo activity of siCD40-SPG complexes was demonstrated as the reduced CD40 protein expression in monocytes and DCs in mice. Furthermore, the in vivo activity of siCD40-SPG targeting human CD40 was confirmed in cynomolgus monkeys by the 5'RLM-RACE technique. In conclusion, we have demonstrated the receptor-ligand binding-mediated delivery of siRNA targeting immune-regulating monocytes and DCs via the interaction of SPG and its receptor, Dectin-1. As monocytes and DCs play central roles in inducing and controlling immune responses, Dectin-1-targeted delivery of nucleic acids should provide a useful tool for developing drugs to treat a wide range of diseases, including autoimmune diseases, allergy, and cancer, as well as transplantation.

Keywords:  Antigen-presenting cell; CD40; Dectin-1; Nucleic acid delivery; Schizophyllan; Targeted delivery; siRNA

DOI:  https://doi.org/10.1016/j.jconrel.2021.09.011
Mol Ther Nucleic Acids. 2021 Dec 03. 26 81-93

Comparison of promoter, DNA vector, and cationic carrier for efficient transfection of hMSCs from multiple donors and tissue sources.

Tyler Kozisek, Andrew Hamann, Luke Samuelson, Miguel Fudolig, Angela K Pannier.

  Human mesenchymal stem cells (hMSCs) are primary cells with high clinical relevance that could be enhanced through genetic modification. However, gene delivery, particularly through nonviral routes, is inefficient. To address the shortcomings of nonviral gene delivery to hMSCs, our lab has previously demonstrated that pharmacological "priming" of hMSCs with clinically approved drugs can increase transfection in hMSCs by modulating transfection-induced cytotoxicity. However, even with priming, hMSC transfection remains inefficient for clinical applications. This work takes a complementary approach to addressing the challenges of transfecting hMSCs by systematically investigating key transfection parameters for their effect on transgene expression. Specifically, we investigated two promoters (cytomegalovirus [CMV] and elongation factor 1 alpha), four DNA vectors (plasmid, plasmid with no F1 origin, minicircle, and mini-intronic plasmid), two cationic carriers (Lipofectamine 3000 and Turbofect), and four donors of hMSCs from two tissues (adipose and bone marrow) for efficient hMSC transfection. Following systematic comparison of each variable, we identified adipose-derived hMSCs transfected with mini-intronic plasmids containing the CMV promoter delivered using Lipofectamine 3000 as the parameters that produced the highest transfection levels. The data presented in this work can guide the development of other hMSC transfection systems with the goal of producing clinically relevant, genetically modified hMSCs.

Keywords:  DNA vector; gene delivery; lipofection; nonviral; polymer-mediated gene delivery; screen; stem cells; transfection

DOI:  https://doi.org/10.1016/j.omtn.2021.06.018
ACS Appl Mater Interfaces. 2021 Sep 15.

Dual-Modality Poly-l-histidine Nanoparticles to Deliver Peptide Nucleic Acids and Paclitaxel for In Vivo Cancer Therapy.

Aniket Wahane, Shipra Malik, Kuo-Chih Shih, Ravinder Reddy Gaddam, Chaohao Chen, Yun Liu, Mu-Ping Nieh, Ajit Vikram, Raman Bahal.

  Cationic polymeric nanoformulations have been explored to increase the transfection efficiency of small molecules and nucleic acid-based drugs. However, an excessive positive charge density often leads to severe cell and tissue-based toxicity that restricts the clinical translation of cationic polymeric nanoformulations. Herein, we investigate a series of cationic poly(lactic-co-glycolic acid) (PLGA)-histidine-based nanoformulations for enhanced cytoplasmic delivery with minimal toxicity. PLGA/poly-l-histidine nanoparticles show promising physico-biochemical features and transfection efficiency in a series of in vitro and cell culture-based studies. Further, the use of acetone/dichloromethane as a solvent mixture during the formulation process significantly improves the morphology and size distribution of PLGA/poly-l-histidine nanoparticles. PLGA/poly-l-histidine nanoformulations undergo clathrin-mediated endocytosis. A contrast-matched small-angle neutron scattering experiment confirmed poly-l-histidine's distribution on the PLGA nanoformulations. PLGA/poly-l-histidine formulations containing paclitaxel as a small molecule-based drug and peptide nucleic acids targeting microRNA-155 as nucleic acid analog are efficacious in in vitro and in vivo studies. PLGA/poly-l-histidine NPs significantly decrease tumor growth in PNA-155 (∼6 fold) and paclitaxel (∼6.5 fold) treatment groups in a lymphoma cell line derived xenograft mice model without inducing any toxicity. Hence, PLGA/poly-l-histidine nanoformulations exhibit substantial transfection efficiency and are safe to deliver reagents ranging from small molecules to synthetic nucleic acid analogs and can serve as a novel platform for drug delivery.

Keywords:  PLGA; microRNAs; nanoparticles; poly-l-histidine; proton-sponge effect

DOI:  https://doi.org/10.1021/acsami.1c11981
Transl Oncol. 2021 Sep 10. pii: S1936-5233(21)00202-3. [Epub ahead of print]14(12): 101210

Radiosensitizing effect of gold nanoparticle loaded with small interfering RNA-SP1 on lung cancer: AuNPs-si-SP1 regulates GZMB for radiosensitivity.

Ming Zhuang, Shan Jiang, Anxin Gu, Xuesong Chen, Mingyan E.

  Radioresistance is a major challenge that largely limits the efficacy of radiotherapy in lung cancer. Gold nanoparticles (AuNPs) are emerging as novel radiosensitizers for cancer patients. Therefore, this study was designed to explore the radiosensitizing effect and mechanism of AuNPs loaded with small interfering RNA (siRNA)-SP1 (AuNPs-si-SP1) on lung cancer. AuNPs-si-SP1 was prepared by the noncovalent binding between AuNPs and siRNA-SP1. The adsorption capacity of AuNPs to siRNA-SP1 was analyzed by gel electrophoresis. The cell uptake of AuNPs-si-SP1 was observed under a laser confocal microscopy. Silencing efficacy of AuNPs-si-SP1 was validated by RT-qPCR and Western blot analysis. Cell viability was determined by CCK-8 assay, radiosensitization by plate colony formation assay, cell apoptosis and cell cycle by flow cytometry, and DNA double strand breaks by immunofluorescence in the presence or absence of AuNPs-si-SP1 or GZMB. The downstream mechanism of SP1 was predicted by bioinformatics analysis, followed by verification by Western blot analysis. Subcutaneous tumorigenesis in nude mice was established to verify the radiosensitization of AuNPs-si-SP1 and GZMB in vivo. AuNPs-si-SP1 effectively absorbed SP1 siRNA and was highly internalized by A549 cells to reduce SP1 protein expression. AuNPs-si-SP1 or GZMB overexpression promoted cells to G2/M phase, DNA double strand breaks, and enhanced radiosensitivity. SP1 could repress GZMB expression in lung cancer cells. In vivo experiments manifested that AuNPs-si-SP1 could inhibit the growth of solid tumor in nude mice to achieve radiosensitization by inhibiting SP1 to upregulate GZMB. AuNPs-si-SP1 might increase the radiosensitivity of lung cancer by inhibiting SP1 to upregulate GZMB.

Keywords:  AuNPs; GZMB; Lung cancer; SP1; siRNA-SP1

DOI:  https://doi.org/10.1016/j.tranon.2021.101210
Small. 2021 Sep 12. e2102494

Reduction-Sensitive Fluorinated-Pt(IV) Universal Transfection Nanoplatform Facilitating CT45-Targeted CRISPR/dCas9 Activation for Synergistic and Individualized Treatment of Ovarian Cancer.

Hongtong Lu, Qingfei Zhang, Shasha He, Sha Liu, Zhigang Xie, Xiaoyuan Li, Yubin Huang.

  Compared to traditional clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, CRISPR/dead Cas9 (dCas9) system can precisely regulate endogenous gene expression without damaging the host gene, representing a greater potential for cancer therapy. Cancer/testis antigen 45 (CT45) is proved to enhance platinum-based chemosensitivity for individualized ovarian cancer therapy. However, the development of a single nanocarrier codelivering CRISPR/dCas9 system and chemotherapeutics for synergistic cancer therapy still faces challenges. Herein, a reduction-sensitive fluorinated-Pt(IV) universal transfection nanoplatform (PtUTP-F) is developed for the CT45-targeted CRISPR/dCas9 activation to achieve synergistic and individualized treatment of ovarian cancer. Overcoming multiple physiological barriers, PtUTP-F condensed gene can efficiently transfect into different cells including 293T cells, A2780, SKOV3, A549, and A2780/cisplatin (DDP) cancer cells, which is superior to Lipofectamine 6000. With the responsive release of gene and Pt(II) in the intracellular reducing microenvironment, PtUTP-F/dCas9-CT45 can generate CRISPR/dCas9 activation of CT45 expression for protein phosphatase 4C (PP4C) activity inhibition to hinder the DNA repair pathway and thus enhances the sensitivity to Pt(II) drugs for individualized A2780 tumor therapy. The PtUTP-F not only represents a powerful nanoplatform for CRISPR/dCas9 system delivery but also initiates a novel strategy for synergistic and individualized treatment of CRISPR/dCas9-based gene therapy with chemotherapy.

Keywords:  CRISPR/dCas9; CT45; individualized treatment; platinum drug; synergistic treatment

DOI:  https://doi.org/10.1002/smll.202102494
Angew Chem Int Ed Engl. 2021 Sep 17.

Supramolecular self-assembled DNA nanosystem for synergistic chemical and gene regulations on cancer cells.

Feng Li, Zhaoyue Lv, Xue Zhang, Yuhang Dong, Xiaohui Ding, Zhemian Li, Shuai Li, Chi Yao, Dayong Yang.

  Incorporating multiple molecular interactions within a system to realize the metabolic reprogramming of cancer cells is prospected to be of great potential in cancer therapy. Herein, we report a supramolecular self-assembled DNA nanosystem, which reprogrammed the cellular antioxidant system via synergistic chemical and gene regulations. In the nanosystem, amphipathic telluroether was coordinated with Mn(II) to self-assemble into micelle, on which a siNrf2 integrated DNA network was assembled. The great electron-donating capability of telluroether was revealed to greatly promote Mn(II)-based Fenton-like reaction to generate subversive ·OH in cancer cells. In response to adenosine triphosphoric acid, the siNrf2 was specially released in cytoplasm for down-regulating expression of detoxification enzymes, which enhanced chemocatalysis-mediated oxidative stress in cancer cells, thus significantly suppressing tumor progression.

Keywords:  DNA nanostructure; DNA nanotechnology; gene therapy; nanomedicine; supramolecular self-assembly

DOI:  https://doi.org/10.1002/anie.202111900
Cancer Cell Int. 2021 Sep 14. 21(1): 485

Engineered exosomes loaded with miR-449a selectively inhibit the growth of homologous non-small cell lung cancer.

Wen Zhou, Mingming Xu, Zhipeng Wang, Mingjun Yang.

  As an efficient drug carrier, exosome has been widely used in the delivery of genetic drugs, chemotherapeutic drugs, and anti-inflammatory drugs. As a genetic drug carrier, exosomes are beneficial to improve transfection efficiency and weaken side effects at the same time. Here, we use genetic engineering to prepare engineered exosomes (miR-449a Exo) that can actively deliver miR-449a. It was verified that miR-449a Exo had good homology targeting capacity and was specifically taken up by A549 cells. Moreover, miR-449a Exo had high delivery efficiency of miR-449a in vitro and in vivo. We demonstrated that miR-449a Exo effectively inhibited the proliferation of A549 cells and promoted their apoptosis. In addition, miR-449a Exo was found to control the progression of mouse tumors and prolong their survival in vivo. Our research provides new ideas for exosomes to efficiently and actively load gene drugs, and finds promising methods for the treatment of non-small cell lung cancer.

Keywords:  Exosomes; Homologous targeting; Non-small cell lung cancer; miR-449a

DOI:  https://doi.org/10.1186/s12935-021-02157-7
J Control Release. 2021 Sep 10. pii: S0168-3659(21)00487-9. [Epub ahead of print]338 694-704

Polyplex nanomicelle delivery of self-amplifying RNA vaccine.

Yi-Hao Chang, Mei-Wei Lin, Ming-Chen Chien, Guan-Ming Ke, I-En Wu, Ren-Li Lin, Chin-Yu Lin, Yu-Chen Hu.

  Self-amplifying RNA (SaRNA) is a burgeoning platform that exploits the replication machinery of alphaviruses such as Venezuelan equine encephalitis (VEE) virus or Sindbis virus (SIN). SaRNA has been used for development of human vaccines, but has not been evaluated for porcine vaccine development. Porcine reproductive and respiratory syndrome virus (PRRSV) causes tremendous economic losses to the worldwide pork industry, but current vaccines trigger delayed neutralizing antibody response and confer only partial protection. Here we first compared two SaRNA systems based on VEE and SIN, and demonstrated that in vitro transcribed VEE-based SaRNA conferred prolonged reporter gene expression and RNA amplification in pig cells with low cytotoxicity, but SIN-based SaRNA imparted evident cytotoxicity and limited gene expression in pig cells. Transfection of VEE-based SaRNA that encodes the major PRRSV antigen dNGP5 (SaRNA-dNGP5) conferred persistent expression for at least 28 days in pig cells. We next complexed SaRNA-dNGP5 with the polyaspartamide block copolymer PEG-PAsp(TEP) to form polyplex nanomicelle with high packaging efficiency and narrow size distribution. The polyplex nanomicelle enabled sustained dNGP5 expression and secretion in vitro. Compared with the commercial PRRS vaccine, nanomicelle delivery of SaRNA-dNGP5 into animal models accelerated the induction of potent neutralizing antibodies with minimal side effects, and elicited stronger IL-4 and IFN-γ responses against homologous and heterologous PRRSV. These properties tackle the problems of current vaccines and implicate the potential of SaRNA-dNGP5 nanomicelle as an effective PRRS vaccine.

Keywords:  Neutralizing antibody; PRRSV; Polyplex nanomicelle; Self-amplifying RNA; Vaccine

DOI:  https://doi.org/10.1016/j.jconrel.2021.09.008
FEBS Open Bio. 2021 Sep 12.

Optimized two-step electroporation process to achieve efficient non-viral-mediated gene insertion into primary T cells.

Ming Yang, Diane Tkach, Alex Boyne, Selena Kazancioglu, Aymeric Duclert, Laurent Poirot, Philippe Duchateau, Alexandre Juillerat.

  The development of gene-editing technologies over the past years has allowed the precise and efficient insertion of transgenes into the genome of various cell types. Knock-in approaches using homology-directed repair and designer nucleases often rely on viral vectors, which can considerably impact the manufacturing cost and timeline of gene-edited therapeutic products. An attractive alternative would be to use naked DNA as a repair template. However, such a strategy faces challenges such as cytotoxicity from double-stranded DNA (dsDNA) to primary cells. Here, we sought to study the kinetics of transcription activator-like effector nuclease (TALEN)-mediated gene editing in primary T cells to improve non-viral gene knock-in. Harnessing this knowledge, we developed a rapid and efficient gene-insertion strategy based on either short single-stranded oligonucleotides or large (2 Kb) linear naked dsDNA sequences. We demonstrated that a time-controlled two-step transfection protocol can substantially improve the efficiency of non-viral transgene integration in primary T cells. Using this approach, we achieved modification of up to ~30% of T cells when inserting a chimeric antigen receptor (CAR) at the T cell receptor alpha constant region (TRAC) locus to generate 'off-the shelf' CAR-T cells.

Keywords:  Double Strand Break; Gene editing; Non-viral vectorization; TALEN; chimeric antigen receptors

DOI:  https://doi.org/10.1002/2211-5463.13292