scholarly journals Versatile Redox-Responsive Polyplexes for the Delivery of Plasmid DNA, Messenger RNA, and CRISPR-Cas9 Genome-Editing Machinery

2018 ◽  
Vol 10 (38) ◽  
pp. 31915-31927 ◽  
Author(s):  
Yuyuan Wang ◽  
Ben Ma ◽  
Amr A. Abdeen ◽  
Guojun Chen ◽  
Ruosen Xie ◽  
...  
Keyword(s):  
Genome Editing ◽  
Plasmid Dna ◽  
Messenger Rna ◽  
Redox Responsive

scholarly journals Nanomedicines to Deliver mRNA: State of the Art and Future Perspectives

Nanomaterials ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 364 ◽  
Author(s):  
Itziar Gómez-Aguado ◽  
Julen Rodríguez-Castejón ◽  
Mónica Vicente-Pascual ◽  
Alicia Rodríguez-Gascón ◽  
María Ángeles Solinís ◽  
...  
Keyword(s):  
Immune Responses ◽  
Plasmid Dna ◽  
Messenger Rna ◽  
Insertional Mutagenesis ◽  
Gene Editing ◽  
State Of The Art ◽  
Translational Efficiency ◽  
Future Perspectives ◽  
Therapeutic Tool

The use of messenger RNA (mRNA) in gene therapy is increasing in recent years, due to its unique features compared to plasmid DNA: Transient expression, no need to enter into the nucleus and no risk of insertional mutagenesis. Nevertheless, the clinical application of mRNA as a therapeutic tool is limited by its instability and ability to activate immune responses; hence, mRNA chemical modifications together with the design of suitable vehicles result essential. This manuscript includes a revision of the strategies employed to enhance in vitro transcribed (IVT) mRNA functionality and efficacy, including the optimization of its stability and translational efficiency, as well as the regulation of its immunostimulatory properties. An overview of the nanosystems designed to protect the mRNA and to overcome the intra and extracellular barriers for successful delivery is also included. Finally, the present and future applications of mRNA nanomedicines for immunization against infectious diseases and cancer, protein replacement, gene editing, and regenerative medicine are highlighted.

scholarly journals Robust genome editing in adult vascular endothelium by nanoparticle delivery of CRISPR-Cas9 plasmid DNA

Cell Reports ◽  
2022 ◽  
Vol 38 (1) ◽  
pp. 110196
Author(s):  
Xianming Zhang ◽  
Hua Jin ◽  
Xiaojia Huang ◽  
Birendra Chaurasiya ◽  
Daoyin Dong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Vascular Endothelium ◽  
Plasmid Dna ◽  
Nanoparticle Delivery

scholarly journals Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3

2021 ◽  
Vol 118 (10) ◽  
pp. e2020401118
Author(s):  
Min Qiu ◽  
Zachary Glass ◽  
Jinjin Chen ◽  
Mary Haas ◽  
Xin Jin ◽  
...  
Keyword(s):  
Genome Editing ◽  
Messenger Rna ◽  
Lipoprotein Metabolism ◽  
Density Lipoprotein ◽  
Blood Lipid ◽  
Loss Of Function ◽  
Guide Rna ◽  
Lipid Nanoparticle ◽  
Delivery Platform

Loss-of-function mutations in Angiopoietin-like 3 (Angptl3) are associated with lowered blood lipid levels, making Angptl3 an attractive therapeutic target for the treatment of human lipoprotein metabolism disorders. In this study, we developed a lipid nanoparticle delivery platform carrying Cas9 messenger RNA (mRNA) and guide RNA for CRISPR-Cas9–based genome editing of Angptl3 in vivo. This system mediated specific and efficient Angptl3 gene knockdown in the liver of wild-type C57BL/6 mice, resulting in profound reductions in serum ANGPTL3 protein, low density lipoprotein cholesterol, and triglyceride levels. Our delivery platform is significantly more efficient than the FDA-approved MC-3 LNP, the current gold standard. No evidence of off-target mutagenesis was detected at any of the nine top-predicted sites, and no evidence of toxicity was detected in the liver. Importantly, the therapeutic effect of genome editing was stable for at least 100 d after a single dose administration. This study highlights the potential of LNP-mediated delivery as a specific, effective, and safe platform for Cas9-based therapeutics.

scholarly journals Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins

2019 ◽  
Author(s):  
Jacob Lamberth ◽  
Laura Daley ◽  
Pachai Natarajan ◽  
Stanislav Khoruzhenko ◽  
Nurit Becker ◽  
...  
Keyword(s):  
Cell Culture ◽  
Genome Editing ◽  
Plasmid Dna ◽  
High Efficiency ◽  
High Specificity ◽  
Human Cells ◽  
Dna And Rna ◽  
Genome Modification ◽  
Delivery Technology ◽  
Target Effects

ABSTRACTCRISPR technology has opened up many diverse genome editing possibilities in human somatic cells, but has been limited in the therapeutic realm by both potential off-target effects and low genome modification efficiencies. Recent advancements to combat these limitations include delivering Cas9 nucleases directly to cells as highly purified ribonucleoproteins (RNPs) instead of the conventional plasmid DNA and RNA-based approaches. Here, we extend RNP-based delivery in cell culture to a less characterized CRISPR format which implements paired Cas9 nickases. The use of paired nickase Cas9 RNP system, combined with a GMP-compliant non-viral delivery technology, enables editing in human cells with high specificity and high efficiency, a development that opens up the technology for further exploration into a more therapeutic role.

Abstract 528: Towards Therapeutic Genome Editing in Vascular Endothelium by Novel Nanoparticle Delivery of Crispr Plasmid Dna

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Xianming Zhang ◽  
You-yang Zhao
Keyword(s):  
Protein Expression ◽  
Genome Editing ◽  
Vascular Endothelium ◽  
Plasmid Dna ◽  
Adult Life ◽  
Therapeutic Delivery ◽  
Crispr System ◽  
Highly Efficient ◽  
Adult Mice

Introduction: Therapeutic delivery of CRISPR system components to induce in vivo genome editing in postnatal and adult life has great translational potential. Recent studies employing non-viral delivery of small guide RNA (gRNA) and Cas9 mRNA have achieved efficient genome editing in adult mice. However, as often seen in other RNA therapeutic studies with non-viral delivery of antisense and siRNA, the efficiency is limited to the liver. Hypothesis: Novel nanoparticle can therapeutically deliver the CRISPR system to selectively target cardiovascular endothelium in adult mice. Methods: We developed novel PLGA-based nanoparticles which was for the first time shown to be uptaken efficiently by the vascular endothelium without specific liver accumulation following i.v. administration. Mixture of the nanoparticle:plasmid DNA expressing Cas9 under the control of the human CDH5 promoter (EC-specific) and gRNA by the U6 promoter was administered i.v. to adult mice. Seven to ten days later, various organ tissues were collected for analysis of the efficiency of genomic editing and knockout of protein expression. The phenotype of CRISPR-mediated in vivo knockout of Pik3cg which encodes the G protein-coupled receptor-activated p110gamma isoform of PI3K was compared to Pik3cg null mice in response to sepsis challenge. Results: Therapeutic delivery of nanoparticles loaded with the all-in-one CRISPR plasmid DNA induced highly efficient genome editing in endothelial cells (ECs) of the cardiovascular system including heart, lung, and aorta in adult mice. The Indel rate was as great as 50% in ECs isolated from these vascular beds. Immunostaining and Western blotting demonstrated greater than 70% decrease of protein expression in ECs. Pik3cg -gRNA-induced genome editing diminished p110γPI3K expression in pulmonary vascular ECs, which led to impaired vascular repair and resolution of inflammation after sepsis challenge as seen in Pik3cg -/- mice. Conclusion: We have developed a simple and highly efficient method for in vivo genome editing selectively targeting the vascular endothelium. This strategy will greatly facilitate cardiovascular research and may enable therapeutic genome editing for prevention and treatment of cardiovascular diseases.

scholarly journals Fluorescence‐Based Quantification of Messenger RNA and Plasmid DNA Decay Kinetics in Extracellular Biological Fluids and Cell Extracts

2020 ◽  
Vol 4 (5) ◽  
pp. 2000057 ◽  
Author(s):  
Heyang Zhang ◽  
Koen Rombouts ◽  
Laurens Raes ◽  
Ranhua Xiong ◽  
Stefaan C. De Smedt ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Messenger Rna ◽  
Biological Fluids ◽  
Decay Kinetics ◽  
Cell Extracts
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