Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells

2021 ◽  
Author(s):  
Pardis Moradi ◽  
akbar hasanzadeh ◽  
Fatemh Radmanesh ◽  
Saideh Rajai Daryasarei ◽  
Elaheh Sadat Hosseini ◽  
...  
Keyword(s):  
Gene Editing ◽  
Transfection Efficiency ◽  
Target Cells ◽  
Reporter Cell ◽  
Expression Plasmid ◽  
Safe Delivery ◽  
Gfp Reporter ◽  
Local Brain ◽  
Transfection Agent

Abstract An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with LMW PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8k in the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5 to 10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g., plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid and in vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.

scholarly journals Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide

2018 ◽  
Vol 115 (19) ◽  
pp. 4903-4908 ◽  
Author(s):  
Hong-Xia Wang ◽  
Ziyuan Song ◽  
Yeh-Hsing Lao ◽  
Xin Xu ◽  
Jing Gong ◽  
...  
Keyword(s):  
Gene Editing ◽  
Transfection Efficiency ◽  
Gene Activation ◽  
Cell Types ◽  
Biological Research ◽  
Knock Out ◽  
Safe Delivery ◽  
Pegylated Nanoparticles

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system

Gene Therapy ◽  
2021 ◽  
Author(s):  
Jonathan O’Keeffe Ahern ◽  
Irene Lara-Sáez ◽  
Dezhong Zhou ◽  
Rodolfo Murillas ◽  
Jose Bonafont ◽  
...  
Keyword(s):  
Delivery System ◽  
Gene Editing ◽  
Transfection Efficiency ◽  
Attractive Alternative ◽  
Safe Delivery ◽  
Guide Rna ◽  
Recessive Dystrophic Epidermolysis Bullosa ◽  
Targeted Gene Editing ◽  
Genomic Editing ◽  
Polymeric Vectors

AbstractRecent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

Delivery of miRNA Using Fe2O3 Nanoparticles Capped Polyethyleneimine as a Nonviral Carrier

2012 ◽  
Vol 531-532 ◽  
pp. 543-546
Author(s):  
Gao Feng Liang ◽  
Ping Li ◽  
Wan Jun Lei
Keyword(s):  
Delivery System ◽  
Hepg2 Cells ◽  
Transfection Efficiency ◽  
Weight Ratio ◽  
Cationic Liposome ◽  
Expression Plasmid ◽  
Safe Delivery ◽  
Rna Interfering ◽  
Dna Complex ◽  
Gfp Tag

An efficient and safe delivery system of RNA interfering is required for clinical application of gene therapy. The study aimed to develop Fe2O3-based nanoparticles for gene delivery to overcome the disadvantages of polyethyleneimine (PEI) or cationic liposome as gene carrier including the cytotoxicity caused by positive charge and aggregation in the cells surface. PEI-capped Fe2O3 nanoparticles are successfully manufactured utilizing Fe2O3 as core, PEI as carapace, which bind miRNA at an appropriate weight ratio by electrostatic interaction and result in well-dispersed nanoparticles. The synthesized GFP tag with miR-26a expression plasmid was used for monitoring transfection efficiency in HepG2 cells. The nanocomplex exhibited higher transfection efficiency and lower cytotoxicity in HepG2 cells than the PEI/DNA complex and commercially available liposome. The delivery resulted in a significantly upregulation of miR-26a in HepG2 cells. Our results offer an alternate delivery system for RNA interfering that can be used on any gene of interest.

A New Protocol for Efficient and Non-Toxic Transfection of siRNA Into Leukemic Cell Lines and Primary Cells – a Direct Comparison Between Nucleofection and Accell Delivery Using Stem Cell Related Antigens As Validation Tools

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4802-4802
Author(s):  
Hanne Østergaard Larsen ◽  
Anne Stidsholt Roug ◽  
Claus Svane Sondergaard ◽  
Peter Hokland
Keyword(s):  
Cell Lines ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Transfection Efficiency ◽  
Leukemic Cell ◽  
Housekeeping Gene ◽  
Target Cells ◽  
Primary Cells ◽  
Cyclophilin B

Abstract Abstract 4802 BACKGROUND: Transient downregulation of genes in vitro by use of siRNA is a time-honored approach to study gene function. This can readily be applied in haematological malignancies, where many lesions derive from altered RNA activity arising e.g. from balanced translocations. PROBLEM FORMULATION: A crucial requirement to obtain this downregulation is an efficient and non-toxic delivery of the siRNA into the target cells, which has proven difficult to accomplish with some protocols; in particular, cells in suspension, such as leukemic blasts. HYPOTHESIS: Directly comparing the range of new transfection methodologies to unravel protocols suitable for cells in suspension. RESULTS: From pilot data, where a range of other methods were excluded due to either low transfection efficiencies or low viability of transfected cells, we compared the gold standard Nucleofection methodology to the novel non-viral based delivery method Accell in both suspension cell lines as well as primary patient AML and CML cells. As control the housekeeping gene Cyclophilin B was used, while hMICL and CD96, recently suggested to be associated with the putative leukemic stem cells, were chosen as target genes. Comparison of the two transfection methods yielded superior results using Accell methodology, as this technique not only yielded higher transfection rates but also retained superior cell viabilities for both cell lines and primary cells. Three AML and two CML samples were included in this study for the validation of transfection efficiency in primary cell employing the programs T-20 and U-15 for Nucleofection. Here, there was a large span in viabilities pre-experimentation among the five patient samples included in this study, probably reflecting the conditions of the patients. However, following transfection there was a significantly lower viability after Nucleofection (mean 37%, range 23%-65%) compared to Accell (mean 82%, range 57%-95%), in the two groups (p<0.001). Corroborating the cell line data transfection efficiencies were clearly superior after Accell transfection of primary patient cells (mean 85%, range 71%-97%) compared to Nucleofection transfection (mean 38%, range 23%-65%), p<0.01. Transfection efficiencies in primary cells after Accell delivery was 90–97% compared to 25–30% using Nucleofection for siRNA delivery. As a functional correlate to these data we performed CFU-GM assays on selected hMICL+ AML patients exposed to anti-hMICL siRNAs. While the AML blast growth of some patients were unaffected by the siRNA treatment others showed a clear inhibition of blast colony formation, suggesting a differential role of hMICL between AML patients, which needs to be further elucidated. CONCLUSION: Accell delivery is thus the method of choice for non-viral transfection of cells in suspension including primary AML and CML cells. These data should provide a platform for further studies of genes involved in early leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Charles A. Lissandrello ◽  
Jose A. Santos ◽  
Peter Hsi ◽  
Michaela Welch ◽  
Vienna L. Mott ◽  
...  
Keyword(s):  
T Cells ◽  
High Throughput ◽  
Continuous Flow ◽  
Viral Vectors ◽  
Large Scale ◽  
Gene Editing ◽  
Cellular Therapy ◽  
Transfection Efficiency ◽  
Target Cells ◽  
Human T Cells

Abstract Implementation of gene editing technologies such as CRISPR/Cas9 in the manufacture of novel cell-based therapeutics has the potential to enable highly-targeted, stable, and persistent genome modifications without the use of viral vectors. Electroporation has emerged as a preferred method for delivering gene-editing machinery to target cells, but a major challenge remaining is that most commercial electroporation machines are built for research and process development rather than for large-scale, automated cellular therapy manufacturing. Here we present a microfluidic continuous-flow electrotransfection device designed for precise, consistent, and high-throughput genetic modification of target cells in cellular therapy manufacturing applications. We optimized our device for delivery of mRNA into primary human T cells and demonstrated up to 95% transfection efficiency with minimum impact on cell viability and expansion potential. We additionally demonstrated processing of samples comprising up to 500 million T cells at a rate of 20 million cells/min. We anticipate that our device will help to streamline the production of autologous therapies requiring on the order of 10$$^8$$ 8 –10$$^9$$ 9 cells, and that it is well-suited to scale for production of trillions of cells to support emerging allogeneic therapies.

scholarly journals Cationic Polymer Nanoparticles-Mediated Delivery of miR-124 Impairs Tumorigenicity of Prostate Cancer Cells

2020 ◽  
Vol 21 (3) ◽  
pp. 869 ◽  
Author(s):  
Raffaele Conte ◽  
Anna Valentino ◽  
Francesca Di Cristo ◽  
Gianfranco Peluso ◽  
Pierfrancesco Cerruti ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Tumor Development ◽  
Transcriptional Level ◽  
Safe Delivery ◽  
Expression Of Genes ◽  
Pc3 Cells ◽  
Delivery Efficiency ◽  
Transfection Agent

MicroRNAs (miRNAs) play a pivotal role in regulating the expression of genes involved in tumor development, invasion, and metastasis. In particular, microRNA-124 (miR-124) modulates the expression of carnitine palmitoyltransferase 1A (CPT1A) at the post-transcriptional level, impairing the ability of androgen-independent prostate cancer (PC3) cells to completely metabolize lipid substrates. However, the clinical translation of miRNAs requires the development of effective and safe delivery systems able to protect nucleic acids from degradation. Herein, biodegradable polyethyleneimine-functionalized polyhydroxybutyrate nanoparticles (PHB-PEI NPs) were prepared by aminolysis and used as cationic non-viral vectors to complex and deliver miR-124 in PC3 cells. Notably, the PHB-PEI NPs/miRNA complex effectively protected miR-124 from RNAse degradation, resulting in a 30% increase in delivery efficiency in PC3 cells compared to a commercial transfection agent (Lipofectamine RNAiMAX). Furthermore, the NPs-delivered miR-124 successfully impaired hallmarks of tumorigenicity, such as cell proliferation, motility, and colony formation, through CPT1A modulation. These results demonstrate that the use of PHB-PEI NPs represents a suitable and convenient strategy to develop novel nanomaterials with excellent biocompatibility and high transfection efficiency for cancer therapy.

scholarly journals Optimization of Synthesis of the Amino Lipid ECO for Effective Delivery of Nucleic Acids

2021 ◽  
Vol 14 (10) ◽  
pp. 1016
Author(s):  
Andrew L. Schilb ◽  
Josef H. Scheidt ◽  
Amita M. Vaidya ◽  
Zhanhu Sun ◽  
Da Sun ◽  
...  
Keyword(s):  
Quality Control ◽  
Nucleic Acids ◽  
Transfection Efficiency ◽  
Solution Phase ◽  
High Yield ◽  
Target Cells ◽  
Synthetic Pathway ◽  
Efficient Delivery ◽  
Size Limitation

Nucleic acids are promising for a variety of therapies, such as cancer therapy and the gene therapy of genetic disorders. The therapeutic efficacy of nucleic acids is reliant on the ability of their efficient delivery to the cytosol of the target cells. Amino lipids have been developed to aid in the cytosolic delivery of nucleic acids. This work reports a new and efficient synthetic pathway for the lipid carrier, (1−aminoethyl) iminobis [N−(oleicylcysteinyl−1−amino−ethyl)propionamide] (ECO). The previous synthesis of the ECO was inefficient and presented poor product quality control. A solution−phase synthesis of the ECO was explored, and each intermediate product was characterized with better quality control. The ECO was synthesized with a relatively high yield and high purity. The formulations of the ECO nanoparticles were made with siRNA, miRNA, or plasmid DNA, and characterized. The transfection efficiency of the nanoparticles was evaluated in vitro over a range of N/P ratios. The nanoparticles were consistent in size with previous formulations and had primarily a positive zeta potential. The ECO/siLuc nanoparticles resulted in potent luciferase silencing with minimal cytotoxicity. The ECO/miR−200c nanoparticles mediated the efficient delivery of miR−200c into the target cells. The ECO/pCMV−GFP nanoparticles resulted in substantial GFP expression upon transfection. These results demonstrate that the solution−phase synthetic pathway produced pure ECO for the efficient intracellular delivery of nucleic acids without size limitation.

scholarly journals Spatial and temporal control of CRISPR/Cas9-mediated gene editing delivered via a light-triggered liposome system

2019 ◽  
Author(s):  
Yagiz Alp Aksoy ◽  
Wenjie Chen ◽  
Ewa M Goldys ◽  
Wei Deng
Keyword(s):  
Gene Editing ◽  
Transfection Efficiency ◽  
Hek293 Cells ◽  
Temporal Control ◽  
Reporter System ◽  
Specific Sequence ◽  
Egfp Gene ◽  
Spatio Temporal

ABSTRACTThe CRISPR-Cas9 and related systems offer a unique genome editing tool allowing facile and efficient introduction of heritable and locus-specific sequence modifications in the genome. Despite its molecular precision, temporal and spatial control of gene editing with CRISPR-Cas9 system is very limited. We developed a light-sensitive liposome delivery system that offers a high degree of spatial and temporal control of gene editing with CRISPR/Cas9 system. We demonstrated its high transfection efficiency, by assessing the targeted knockout of eGFP gene in human HEK293 cells (52.8% knockout). We further validated our results at a single-cell resolution using an in vivo eGFP reporter system in zebrafish (77% knockout). To the best of our knowledge we reported the first proof-of-concept of spatio-temporal control of CRISPR/Cas9 by using light-triggered liposomes in both in vitro and in vivo environment.

In-silico Molecular Interaction of Short Synthetic Lipopeptide/Importin-alpha and In-vitro Evaluation of Transgene Expression Mediated by Liposome- Based Gene Carrier

2020 ◽  
Vol 20 (5) ◽  
pp. 383-394
Author(s):  
Tarwadi Tarwadi ◽  
Jalal A. Jazayeri ◽  
Sabar Pambudi ◽  
Alfan D. Arbianto ◽  
Heni Rachmawati ◽  
...  
Keyword(s):  
Molecular Interaction ◽  
In Silico ◽  
Transgene Expression ◽  
Transfection Efficiency ◽  
Dna Molecules ◽  
In Vitro Evaluation ◽  
Nuclear Uptake ◽  
Importin Α ◽  
Transfection Agent

Background: Lipopeptide-based gene carriers have shown low cytotoxicity, are capable of cell membrane penetration, are easy to manufacture and therefore are great potential candidates for gene delivery applications. Objectives: This study aims to explore a range of short synthetic lipopeptides, (Lau: Lauryl; Pal: Palmitoyl) consisting of an alkyl chain, one cysteine (C), 1 to 2 histidine (H), and lysine (K) residues by performing in-silico molecular interaction and in-vitro evaluation. Methods: The molecular interactions between the lipopeptides and Importin-α receptor were performed using AutoDock Vina and Amber14. The lipopeptide/DNA complexes were evaluated in- -vitro for their interactions, particle size, zeta potential and transgene expression. Transfection efficiency of the lipopeptides and Pal-CKKHH-derived liposome was carried out based on luciferase transgene expression. Results: The in-silico interaction showed that Lau-CKKH and Pal-CKKHH hypothetically expedited nuclear uptake. Both lipopeptides had lower binding energy (-6.3 kcal/mol and -6.2 kcal/mol, respectively), compared to the native ligand, viz, nuclear localization sequence (-5.4 kcal/mol). The short lipopeptides were able to condense DNA molecules and efficiently form compacted nanoparticles. Based on the in-vitro evaluation on COS-7, Pal-CKKHH was found to be the best transfection agent amongst the lipopeptides. Its transfection efficiency (ng Luc/mg total protein) increased up to ~3-fold higher (1163 + 55) as it was formulated with helper lipid DOPE (1:2). The lipopeptide- based liposome (Pal-CKKHH: DOPE=1:2) also facilitated luciferase transgene expression on human embryonic kidney cells (293T) and human cervical adenocarcinoma cells (HeLa) with transfection efficiency 1779 +52 and 260 + 22, respectively. Conclusion: Our study for the first time has shown that the fully synthesized short lipopeptide Pal- CKKHH is able to interact firmly with the Importin-α. The lipopeptide is able to condense DNA molecules efficiently, facilitate transgene expression, expedite the nuclear uptake process, and hence has the characteristics of a potential transfection agent.

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