scholarly journals Nuclear-Targeting Delivery of CRISPRa System for Upregulation of β-Defensin against Virus Infection by Dexamethasone and Phenylalanine Dual-Modified Dendrimer

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Mingxiang Zuo ◽  
Xiaoxia Li ◽  
Shuang Liu ◽  
Bin Chen ◽  
Du Cheng
Keyword(s):  
Virus Infection ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Viral Disease ◽  
Dna Encoding ◽  
Nuclear Targeting ◽  
Guide Rna ◽  
Targeting Delivery ◽  
Vesicular Stomatitis

The dual-modified dendrimer containing dexamethasone (DET) and phenylalanine (Phe) was prepared to deliver plasmid DNA encoding dCas9 and single-guide RNA (sgRNA) for specific upregulation of β-defensin. DET and Phe moieties synergistically enhanced the transfection efficiency and reduced cytotoxicity of dendrimers. Combination of three sgRNAs targeting β-defensin gene demonstrated higher activation efficacy of β-defensin than any single sgRNA and combinations of any two sgRNAs, showing an efficient inhibition of virus infection and replication. The titer of vesicular stomatitis virus (VSV) in the cells treated with dCas9-sgRNA targeting β-defensin was reduced by about 100-fold compared to that of cells treated with dCas9-scramble sgRNA (dCas9-scr sgRNA). In vivo experiments demonstrated that the DET- and Phe-modified dendrimer effectively delivered plasmid DNA encoding dCas9 protein into the airway epithelium, inducing β-defensin expression. Delivery of the CRISPR activation system by a dendrimer modified with DET and Phe was a promising approach against viral disease.

scholarly journals TAMI-14. NANOPARTICLE DELIVERY OF PD-L1 CRISPR/CAS9 PLASMID DNA FOR ANTI-GLIOBLASTOMA IMMUNOTHERAPY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii216-ii216
Author(s):  
Javier Fierro ◽  
An Tran ◽  
Chris Factoriza ◽  
Brandon Chin ◽  
Huanyu Dou
Keyword(s):  
Plasmid Dna ◽  
Viral Vectors ◽  
New Technologies ◽  
Transfection Efficiency ◽  
Guide Rna ◽  
Immune Balance ◽  
Main Challenge ◽  
Low Cytotoxicity

Abstract Glioblastoma multiforme (GBM) is a devastating cancer that develops from astrocytes in the brain. GBM is fast acting and kills 90% of patients within 5 years. Several immunotherapies have been developed to treat GBM, however, major challenges still persist. For example, checkpoint proteins such as programmed cell death protein 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), are upregulated in GBM cells to evade the immune system. Targeting PD-L1 for genetic knockdown is thus a promising avenue for the treatment of GBM. However, PD-L1 protein inhibitors have been shown to cause immune overreaction and toxicity, therefore requiring new technologies. CRISPR/Cas9 gene editing has been widely used for the study and treatment of many diseases, but has not been extensively studied for the treatment of GBM. The main challenge is developing a gene delivery platform for the delivery of CRISPR/Cas9 plasmid DNA (pDNA). Many viral vectors have been used for the delivery of pDNA, but unfortunately are associated with high toxicity. Nanotechnology is emerging as a new platform for the delivery of pDNA as it shows high transfection efficiency with low cytotoxicity. We developed a cationic core-shell nanoparticle (NP) capable of carrying CRISPR/Cas9 pDNA. This plasmid contains multiple guide RNA (gRNA) expression cassettes for the knockdown of PD-L1. PDL1gRNA-CRISPR/Cas9pDNA-NPs were taken up by U87 cells within 30 minutes, and entered into the nucleus at 2 hours. The effective delivery of PDL1gRNA-CRISPR/Cas9pDNA-NPs led to the expression of PD-L1 gRNA and Cas9 enzyme, and the knockdown of PD-L1. Regulation of immune balance was determined after PD-L1 knockdown in vitro and in vivo. Our study shows the potential of NP-based PDL1gRNA-CRISPR/Cas9 delivery as an anti-GBM immunotherapy for clinical applications.

scholarly journals Two Antibody-Guided Lactic-co-Glycolic Acid-Polyethylenimine (LGA-PEI) Nanoparticle Delivery Systems for Therapeutic Nucleic Acids

2021 ◽  
Vol 14 (9) ◽  
pp. 841
Author(s):  
Jian-Ming Lü ◽  
Zhengdong Liang ◽  
Dongliang Liu ◽  
Bin Zhan ◽  
Qizhi Yao ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Plasmid Dna ◽  
Glycolic Acid ◽  
Growth Factor Receptor ◽  
Green Fluorescence Protein ◽  
Active Targeting ◽  
Single Chain ◽  
Targeting Delivery

We previously reported a new polymer, lactic-co-glycolic acid-polyethylenimine (LGA-PEI), as an improved nanoparticle (NP) delivery for therapeutic nucleic acids (TNAs). Here, we further developed two antibody (Ab)-conjugated LGA-PEI NP technologies for active-targeting delivery of TNAs. LGA-PEI was covalently conjugated with a single-chain variable fragment antibody (scFv) against mesothelin (MSLN), a biomarker for pancreatic cancer (PC), or a special Ab fragment crystallizable region-binding peptide (FcBP), which binds to any full Ab (IgG). TNAs used in the current study included tumor suppressor microRNA mimics (miR-198 and miR-520h) and non-coding RNA X-inactive specific transcript (XIST) fragments; green fluorescence protein gene (GFP plasmid DNA) was also used as an example of plasmid DNA. MSLN scFv-LGA-PEI NPs with TNAs significantly improved their binding and internalization in PC cells with high expression of MSLN in vitro and in vivo. Anti-epidermal growth factor receptor (EGFR) monoclonal Ab (Cetuximab) binding to FcBP-LGA-PEI showed active-targeting delivery of TNAs to EGFR-expressing PC cells.

scholarly journals Eradication of Swine Vesicular Disease in Italy

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1269 ◽  
Author(s):  
Marco Tamba ◽  
Francesco Plasmati ◽  
Emiliana Brocchi ◽  
Luigi Ruocco
Keyword(s):  
Virus Infection ◽  
Foot And Mouth Disease ◽  
Viral Disease ◽  
Virus Eradication ◽  
Complete Control ◽  
Testing Program ◽  
Virus Activity ◽  
Pig Farms ◽  
Vesicular Stomatitis ◽  
Vesicular Disease

Swine vesicular disease (SVD) is a contagious viral disease of pigs clinically indistinguishable from other vesicular diseases, such as foot and mouth disease, vesicular stomatitis, vesicular exanthema of swine, and idiopathic vesicular disease. In Italy, where SVD was first reported in 1966, an eradication program started in 1995. The program, updated in 2008, was based on regionalization, complete control on pig movements, improvement of pig farms biosecurity, appropriate cleansing and disinfection procedures of vehicles approved for pig transportation, and a testing program using both serological and virological assays. In cases of confirmed SVD virus infection a stamping-out policy was applied. In the period 2009 to 2019, between 300,000 and 400,000 pigs were serologically tested each year. The last SVD outbreak was notified in 2015, and the last seropositive pig was detected in 2017. SVD surveillance is still ongoing and no proof of virus activity has been detected so far. All available data support the complete SVD virus eradication from the Italian pig industry.

scholarly journals Magnetic field contributes to the cellular uptake for effective therapy with magnetofection using plasmid DNA encoding against Mcam in B16F10 melanoma in vivo

Nanomedicine ◽  
2016 ◽  
Vol 11 (6) ◽  
pp. 627-641 ◽  
Author(s):  
Lara Prosen ◽  
Samo Hudoklin ◽  
Maja Cemazar ◽  
Monika Stimac ◽  
Ursa Lampreht Tratar ◽  
...  
Keyword(s):  
Magnetic Field ◽  
Cellular Uptake ◽  
Plasmid Dna ◽  
Effective Therapy ◽  
Dna Encoding ◽  
B16f10 Melanoma

scholarly journals Dual delivery of nucleic acids and PEGylated-bisphosphonates via calcium phosphate nanoparticles

2019 ◽  
Author(s):  
Sofia Bisso ◽  
Simona Mura ◽  
Bastien Castagner ◽  
Patrick Couvreur ◽  
Jean-Christophe Leroux
Keyword(s):  
Calcium Phosphate ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Physical Stability ◽  
Entrapment Efficiency ◽  
Endosomal Escape ◽  
Particle Uptake ◽  
Success Stories

AbstractDespite many years of research and a few success stories with gene therapeutics, efficient and safe DNA delivery remains a major bottleneck for the clinical translation of gene-based therapies. Gene transfection with calcium phosphate (CaP) nanoparticles brings the advantages of low toxicity, high DNA entrapment efficiency and good endosomal escape properties. The macroscale aggregation of CaP nanoparticles can be easily prevented through surface coating with bisphosphonate conjugates. Bisphosphonates, such as alendronate, recently showed promising anticancer effects. However, their poor cellular permeability and preferential bone accumulation hamper their full application in chemotherapy. Here, we investigated the dual delivery of plasmid DNA and alendronate using CaP nanoparticles, with the goal to facilitate cellular internalization of both compounds and potentially achieve a combined pharmacological effect on the same or different cell lines. A pH-sensitive poly(ethylene glycol)-alendronate conjugate was synthetized and used to formulate stable plasmid DNA-loaded CaP nanoparticles. These particles displayed good transfection efficiency in cancer cells and a strong cytotoxic effect on macrophages. The in vivo transfection efficiency, however, remained low, calling for an improvement of the system, possibly with respect to the extent of particle uptake and their physical stability.Graphical abstract

scholarly journals 3D Printed Gene-activated Octacalcium Phosphate Implants for Large Bone Defects Engineering

2020 ◽  
Vol 6 (3) ◽  
Author(s):  
Ilya I Bozo ◽  
Roman V. Deev ◽  
Igor V. Smirnov ◽  
Alexander Yu Fedotov ◽  
Vladimir K. Popov ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Three Dimensional ◽  
Bone Substitutes ◽  
Octacalcium Phosphate ◽  
Dna Encoding ◽  
X Ray Diffraction ◽  
3D Printed ◽  
Clinical Potential ◽  
Large Bone

The aim of the study was the development of three-dimensional (3D) printed gene-activated implants based on octacalcium phosphate (OCP) and plasmid DNA encoding VEGFA. The first objective of the present work involved design and fabrication of gene-activated bone substitutes based on the OCP and plasmid DNA with VEGFА gene using 3D printing approach of ceramic constructs, providing the control of its architectonics compliance to the initial digital models. X-ray diffraction, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and compressive strength analyses were applied to investigate the chemical composition, microstructure, and mechanical properties of the experimental samples. The biodegradation rate and the efficacy of plasmid DNA delivery in vivo were assessed during standard tests with subcutaneous implantation to rodents in the next stage. The final part of the study involved substitution of segmental tibia and mandibular defects in adult pigs with 3D printed gene-activated implants. Biodegradation, osteointegration, and effectiveness of a reparative osteogenesis were evaluated with computerized tomography, SEM, and a histological examination. The combination of gene therapy and 3D printed implants manifested the significant clinical potential for effective bone regeneration in large/critical size defect cases.

Effects of lyoprotectants on long-term stability and transfection efficacy of lyophilized poly(lactide-co-glycolide)-graft-polyethylenimine/plasmid DNA polyplexes

Nanomedicine ◽  
2021 ◽  
Author(s):  
Joshua Woo ◽  
Jeoung Soo Lee
Keyword(s):  
Plasmid Dna ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Dna Encoding ◽  
Rat Glioma ◽  
Term Stability ◽  
Long Term Stability ◽  
Glioma C6 ◽  
Transfection Efficacy

Aim: We investigated the effect of lyoprotectants on the long-term stability and transfection efficiency of lyophilized (Lyo.) polyplexes prepared from poly(lactide-co-glycolide)-graft-polyethylenimine (PgP) and plasmid DNA encoding green fluorescent protein (pGFP). Materials & methods: Lyo. PgP/pGFP polyplexes prepared with/without lyoprotectants were stored at -20°C over 6 months. Polyplex stability was analyzed by gel electrophoresis and heparin competition assay. Transfection efficiency and cytotoxicity were evaluated in rat glioma (C6) cells in medium containing 10% serum. Results: Lyo. PgP/pGFP polyplexes prepared with 5% sucrose as a lyoprotectant remained stable up to 6 months and retained transfection efficiency up to 4 months. Conclusion: Lyo. PgP-based polyplexes retain bioactivity during extended storage, potentially enabling transport to remote regions and less stable settings, increasing access to life-changing gene therapy.

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