scholarly journals Stabilization of Poly (β-Amino Ester) Nanoparticles for the Efficient Intracellular Delivery of PiggyBac Transposon

2021 ◽  
Vol 8 (2) ◽  
pp. 16
Author(s):  
Tina Rodgers ◽  
Nicolas Muzzio ◽  
Caleb Watson ◽  
Gabriela Romero
Keyword(s):  
Mammalian Cells ◽  
Viral Vectors ◽  
Gene Editing ◽  
Transfection Efficiency ◽  
Release Kinetics ◽  
Green Fluorescence Protein ◽  
Intracellular Delivery ◽  
Amino Ester ◽  
Piggybac Transposon ◽  
Promising Therapeutic Approach

The administration of gene-editing tools has been proposed as a promising therapeutic approach for correcting mutations that cause diseases. Gene-editing tools, composed of relatively large plasmid DNA constructs that often need to be co-delivered with a guiding protein, are unable to spontaneously penetrate mammalian cells. Although viral vectors facilitate DNA delivery, they are restricted by the size of the plasmid to carry. In this work, we describe a strategy for the stable encapsulation of the gene-editing tool piggyBac transposon into Poly (β-amino ester) nanoparticles (NPs). We propose a non-covalent and a covalent strategy for stabilization of the nanoformulation to slow down release kinetics and enhance intracellular delivery. We found that the formulation prepared by covalently crosslinking Poly (β-amino ester) NPs are capable to translocate into the cytoplasm and nuclei of human glioblastoma (U87MG) cells within 1 h of co-culturing, without the need of a targeting moiety. Once internalized, the nanoformulation dissociates, delivering the plasmid presumably as a response to the intracellular acidic pH. Transfection efficiency is confirmed by green fluorescence protein (GFP) expression in U87MG cells. Covalently stabilized Poly (β-amino ester) NPs are able to transfect ~55% of cells causing non-cytotoxic effects. The strategy described in this work may serve for the efficient non-viral delivery of other gene-editing tools.

scholarly journals Graphene Oxide as 2D Platform for Complexation and Intracellular Delivery of siRNA

2018 ◽  
Author(s):  
Irene de Lázaro ◽  
Sandra Vranic ◽  
Domenico Marson ◽  
Artur Filipe Rodrigues ◽  
Maurizio Buggio ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Nucleic Acid ◽  
Gene Silencing ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Unmet Need ◽  
Sharp Decrease ◽  
Intracellular Delivery ◽  
Nucleic Acid Delivery ◽  
Primary Mouse

AbstractThe development of efficient and safe nucleic acid delivery vectors remains an unmet need holding back translation of gene therapy approaches into bedside. Graphene oxide (GO) could help bypass such bottleneck thanks to its large surface area, versatile chemistry and biocompatibility, which could overall enhance transfection efficiency while abolishing some of the limitations linked to the use of viral vectors. Here, we aimed to assess the capacity of bare GO, without any further surface modification, to complex a short double-stranded nucleic acid of biological relevance (siRNA) and mediate its intracellular delivery. GO formed stable complexes with siRNA at 10:1, 20:1 and 50:1 GO:siRNA mass ratios. Complexation was further corroborated by atomistic molecular dynamics simulations. GO:siRNA complexes were promptly internalized in a primary mouse cell culture, as early as 4 h after exposure. At this time point, intracellular siRNA levels were comparable to those provided by a lipid-based transfection reagent that achieved significant gene silencing. Time-lapse tracking of internalized GO and siRNA evidenced a sharp decrease of intracellular siRNA from 4 to 12 h, while GO was sequestered in large vesicles, which may explain the lack of biological effect (i.e. gene silencing) achieved by GO:siRNA complexes. This study underlines the potential of non-surface modified GO flakes to act as 2D siRNA delivery platforms, without the need for cationic functionalization, but warrants further vector optimization to allow effective release of the nucleic acid and achieve efficient gene silencing.

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.

Stepwise Development of Biomimetic Chimeric Peptides for Gene Delivery

2020 ◽  
Vol 27 (8) ◽  
pp. 698-710
Author(s):  
Roya Cheraghi ◽  
Mahboobeh Nazari ◽  
Mohsen Alipour ◽  
Saman Hosseinkhani
Keyword(s):  
Gene Delivery ◽  
Targeted Delivery ◽  
Viral Vectors ◽  
Large Scale ◽  
Transfection Efficiency ◽  
Cationic Lipids ◽  
Target Cells ◽  
Scale Production ◽  
Stepwise Development ◽  
Chimeric Peptides

Gene-based therapy largely relies on the vector type that allows a selective and efficient transfection into the target cells with maximum efficacy and minimal toxicity. Although, genes delivered utilizing modified viruses transfect efficiently and precisely, these vectors can cause severe immunological responses and are potentially carcinogenic. A promising method of overcoming this limitation is the use of non-viral vectors, including cationic lipids, polymers, dendrimers, and peptides, which offer potential routes for compacting DNA for targeted delivery. Although non-viral vectors exhibit reduced transfection efficiency compared to their viral counterpart, their superior biocompatibility, non-immunogenicity and potential for large-scale production make them increasingly attractive for modern therapy. There has been a great deal of interest in the development of biomimetic chimeric peptides. Biomimetic chimeric peptides contain different motifs for gene translocation into the nucleus of the desired cells. They have motifs for gene targeting into the desired cell, condense DNA into nanosize particles, translocate the gene into the nucleus and enhance the release of the particle into the cytoplasm. These carriers were developed in recent years. This review highlights the stepwise development of the biomimetic chimeric peptides currently being used in gene delivery.

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 Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

scholarly journals Bioluminescent Optogenetics: A Novel Experimental Therapy to Promote Axon Regeneration after Peripheral Nerve Injury

2021 ◽  
Vol 22 (13) ◽  
pp. 7217
Author(s):  
Arthur W. English ◽  
Ken Berglund ◽  
Dario Carrasco ◽  
Katharina Goebel ◽  
Robert E. Gross ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Peripheral Nerve ◽  
Nerve Injury ◽  
Peripheral Nerve Injury ◽  
Viral Vectors ◽  
Axon Regeneration ◽  
Intraperitoneal Administration ◽  
Experimental Therapy ◽  
Promising Therapeutic Approach ◽  
Compound Muscle Action Potentials

Functional recovery after peripheral nerve injury (PNI) is poor, mainly due to the slow and incomplete regeneration of injured axons. Experimental therapies that increase the excitability of the injured axons have proven remarkably successful in promoting regeneration, but their clinical applicability has been limited. Bioluminescent optogenetics (BL-OG) uses luminopsins, fusion proteins of light-generating luciferase and light-sensing ion channels that could be used to increase neuronal excitability if exposed to a suitable substrate. Excitatory luminopsins were expressed in motoneurons of transgenic mice and in wildtype mice transduced with adeno-associated viral vectors. Intraperitoneal administration of coelenterazine (CTZ), a known luciferase substrate, generated intense bioluminescence in peripheral axons. This bioluminescence increased motoneuron excitability. A single administration of CTZ immediately after sciatic nerve transection and repair markedly enhanced motor axon regeneration. Compound muscle action potentials were 3–4 times larger than controls by 4 weeks after injury. The results observed with transgenic mice were comparable to those of mice in which the luminopsin was expressed using viral vectors. Significantly more motoneurons had successfully reinnervated muscle targets four weeks after nerve injury in BL-OG treated mice than in controls. Bioluminescent optogenetics is a promising therapeutic approach to enhancing axon regeneration after PNI.

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.

scholarly journals Microfluidic Based Physical Approaches towards Single-Cell Intracellular Delivery and Analysis

Micromachines ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 631
Author(s):  
Kiran Kaladharan ◽  
Ashish Kumar ◽  
Pallavi Gupta ◽  
Kavitha Illath ◽  
Tuhin Subhra Santra ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Biology ◽  
Transfection Efficiency ◽  
Intracellular Delivery ◽  
Diagnostic Tools ◽  
Delivery Methods ◽  
Physical Methods ◽  
Cell Measurement ◽  
Therapeutic Development ◽  
Single Living

The ability to deliver foreign molecules into a single living cell with high transfection efficiency and high cell viability is of great interest in cell biology for applications in therapeutic development, diagnostics, and drug delivery towards personalized medicine. Various physical delivery methods have long demonstrated the ability to deliver cargo molecules directly to the cytoplasm or nucleus and the mechanisms underlying most of the approaches have been extensively investigated. However, most of these techniques are bulk approaches that are cell-specific and have low throughput delivery. In comparison to bulk measurements, single-cell measurement technologies can provide a better understanding of the interactions among molecules, organelles, cells, and the microenvironment, which can aid in the development of therapeutics and diagnostic tools. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great interest. In recent years, single-cell technologies have become increasingly robust and accessible, although limitations exist. This review article aims to cover various microfluidic-based physical methods for single-cell intracellular delivery such as electroporation, mechanoporation, microinjection, sonoporation, optoporation, magnetoporation, and thermoporation and their analysis. The mechanisms of various physical methods, their applications, limitations, and prospects are also elaborated.

scholarly journals A Review on Electroporation-Based Intracellular Delivery

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 3044 ◽  
Author(s):  
Junfeng Shi ◽  
Yifan Ma ◽  
Jing Zhu ◽  
Yuanxin Chen ◽  
Yating Sun ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Safety Issue ◽  
Intracellular Delivery ◽  
Cellular Reprogramming ◽  
Gene Therapies ◽  
Potential Safety ◽  
Potential Applications ◽  
Dose Control ◽  
Intracellular Probe ◽  
New Generation

Intracellular delivery is a critical step in biological discoveries and has been widely utilized in biomedical research. A variety of molecular tools have been developed for cell-based gene therapies, including FDA approved CAR-T immunotherapy, iPSC, cell reprogramming and gene editing. Despite the inspiring results of these applications, intracellular delivery of foreign molecules including nucleic acids and proteins remains challenging. Efficient yet non-invasive delivery of biomolecules in a high-throughput manner has thus long fascinates the scientific community. As one of the most popular non-viral technologies for cell transfection, electroporation has gone through enormous development with the assist of nanotechnology and microfabrication. Emergence of miniatured electroporation system brought up many merits over the weakness of traditional electroporation system, including precise dose control and high cell viability. These new generation of electroporation systems are of considerable importance to expand the biological applications of intracellular delivery, bypassing the potential safety issue of viral vectors. In this review, we will go over the recent progresses in the electroporation-based intracellular delivery and several potential applications of cutting-edge research on the miniatured electroporation, including gene therapy, cellular reprogramming and intracellular probe.

pH-sensing nano-crystals of carbonate apatite: Effects on intracellular delivery and release of DNA for efficient expression into mammalian cells

Gene ◽  
2006 ◽  
Vol 376 (1) ◽  
pp. 87-94 ◽  
Author(s):  
E.H. Chowdhury ◽  
A. Maruyama ◽  
A. Kano ◽  
M. Nagaoka ◽  
M. Kotaka ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Intracellular Delivery ◽  
Carbonate Apatite ◽  
Ph Sensing ◽  
Efficient Expression
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