Gene editing is a versatile technique in biomedicine that promotes fundamental research as well as clinical therapy. The development of Clustered Regularly Interspaced Short Palindromic Repeats ...(CRISPR) as a genome editing machinery has accelerated the application of gene editing. However, the delivery of CRISPR components often suffers when using conventional transfection methods, such as viral transduction and chemical vectors, due to limited packaging size and inefficiency toward certain cell types. In this review, we discuss physical transfection methods for CRISPR gene editing which can overcome these limitations. We outline different types of physical transfection methods, highlight novel techniques to deliver CRISPR components, and emphasize the role of micro and nanotechnology to improve transfection performance. We present our perspectives on the limitations of current technology and provide insights on the future developments of physical transfection methods.
DNA Cage Delivery to Mammalian Cells Walsh, Anthony S; Yin, HaiFang; Erben, Christoph M ...
ACS nano,
07/2011, Letnik:
5, Številka:
7
Journal Article
Recenzirano
DNA cages are nanometer-scale polyhedral structures formed by self-assembly from synthetic DNA oligonucleotides. Potential applications include in vivo imaging and the targeted delivery of ...macromolecules into living cells. We report an investigation of the ability of a model cage, a DNA tetrahedron, to enter live cultured mammalian cells. Cultured human embryonic kidney cells were treated with a range of fluorescently labeled DNA tetrahedra and subsequently examined using confocal microscopy and flow cytometry. Substantial uptake of tetrahedra into cells was observed both when the cells were treated with tetrahedra alone and when the cells were treated with a mixture of tetrahedra and a transfection reagent. Analysis of the subcellular localization of transfected tetrahedra using confocal microscopy and organelle staining indicates that the cages are located in the cytoplasm. FRET experiments indicate that the DNA cages remain substantially intact within the cells for at least 48 h after transfection. This is a first step toward the use of engineered DNA nanostructures to deliver and control the activity of cargoes within cells.
Cryptosporidium parvum can be reliably genetically manipulated using CRISPR/Cas9-driven homologous repair coupled to in vivo propagation within immunodeficient mice. Recent modifications have ...simplified the initial protocol significantly. This chapter will guide through procedures for excystation, transfection, infection, collection, and purification of transgenic Cryptosporidium parvum.
Human mesenchymal stem cells (hMSCs) are being researched for cell-based therapies due to a host of unique properties, however, genetic modification of hMSCs, accomplished through nonviral gene ...delivery, could greatly advance their therapeutic potential. Furthermore, expression of multiple transgenes in hMSCs could greatly advance their clinical significance for treatment of multifaceted diseases, as individual transgenes could be expressed that target separate pathogenic drivers of complex diseases. Expressing multiple transgenes can be accomplished by delivering multiple DNA vectors encoding for each transgene, or by delivering a single poly-cistronic vector that encodes for each transgene and accomplishes expression through either use of multiple promoters, an internal ribosome entry site (IRES), or a 2A peptide sequence. These different transgene expression strategies have been used to express multiple transgenes in various mammalian cells, however, they have not been fully evaluated in difficult-to-transfect primary cells, like hMSCs. This study systematically compared four transgene expression and delivery strategies for expression of two reporter transgenes in four donors of hMSCs from two tissue sources using lipid- and polymer-mediate transfection, as follows: (i) delivery of separate DNA vectors in separate nanoparticles; (ii) delivery of separate DNA vectors combined in the same nanoparticle; (iii) delivery of a bi-cistronic DNA vector with an IRES sequence via nanoparticles; and (iv) delivery of a bi-cistronic DNA vector with a dual 2A peptide sequence via nanoparticles.
Our results indicate that expression of two transgenes in hMSCs, independent of expression or delivery strategy, is inefficient compared to expressing a single transgene. However, delivery of separate DNA vectors complexed in the same nanoparticle, or delivery of a bi-cistronic DNA vector with a dual 2A peptide sequence, significantly increased the number of hMSCs expressing both transgenes compared to other conditions tested.
Separate DNA vectors delivered in the same nanoparticle and bi-cistronic DNA vectors with dual 2A peptide sequences are highly efficient at simultaneously expressing two transgenes in multiple donors of hMSCs from different tissue sources. The data presented in this work can guide the development of hMSC transfection systems for delivery of multiple transgenes, with the goal of producing clinically relevant, genetically modified hMSCs.
The specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window ...for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.
The red fluorescent protein(rfp)-blasticidin deaminase(bsd) fusion gene was transfected into Babesia ovata by electroporation with the plasmid DNA and selected with 15 μg/mL of blasticidin S under ...the in vitro culture condition. The transfected parasite with episomal DNA was selected and cultured for further analysis based on the presence of the rfp-bsd fusion gene by PCR and expression of the fusion protein by immunofluorescence antibody test under fluorescence microscopy for 2 months after the transfection. The results are the first, to our knowledge, to demonstrate the expression and stability of the episomal rfp-bsd fusion gene under the control of actin promoter as a selectable marker for the transfection system in B. ovata.
The concept of PLGA-lipid hybrid nanoparticles arose as a motivation for developing drug delivery systems with versatile physical and chemical properties, unequivocal therapeutic benefits and ...minimized side effects. These formulations opened a new avenue of “smart tools” in the development of personalized, noninvasive emerging nanomedicine. However, there is still the risk that smart hybrid system manufactured from a mixture of materials, such as PLGA in combination with synthetic/vegetable lipids and targeting agents might trigger high nanotoxicological responses. This review is focused on the detailed description of PLGA-lipids/oils hybridization concept and its effect on the performances of nanocarriers as powerful, versatile delivery system, the manufacturing methods, the main applications, and their potential clinical impact. The successful design of a hybrid formulation involves two foremost challenges, firstly a comprehensive consideration of essential characteristics of each constituent that form the hybrid system and secondly, a deep understanding of the interactions of hybrid nanocarrier with cells in order to predict the potential toxicity issues and ensure its safe clinical applications.
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•PLGA-based nanocarriers represent ones of the most versatile delivery system used in the field of personalized nanomedicine.•The combination of PLGA-based nanoparticles with graphene derivatives is a promising platform for theranostic applications.•The successful design of a hybrid carrier requires an ample study of the physicochemical properties of each constituent.•Targeting and release mechanisms need to be clearly debriefed to warranty drug stability, biosafety, therapeutic efficiency.
The original version of this Article had an incorrect Article number of 1, an incorrect Volume of 5 and an incorrect Publication year of 2019. These errors have now been corrected in the PDF and HTML ...versions of the Article.
Nucleic acids carry the building plans of living systems. As such, they can be exploited to make cells produce a desired protein, or to shut down the expression of endogenous genes or even to repair ...defective genes. Hence, nucleic acids are unique substances for research and therapy. To exploit their potential, they need to be delivered into cells which can be a challenging task in many respects. During the last decade, nanomagnetic methods for delivering and targeting nucleic acids have been developed, methods which are often referred to as magnetofection. In this review we summarize the progress and achievements in this field of research. We discuss magnetic formulations of vectors for nucleic acid delivery and their characterization, mechanisms of magnetofection, and the application of magnetofection in viral and nonviral nucleic acid delivery in cell culture and in animal models. We summarize results that have been obtained with using magnetofection in basic research and in preclinical animal models. Finally, we describe some of our recent work and end with some conclusions and perspectives.
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Formulations based on ionizable amino‐lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, ...in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre‐clinical translation. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3R guidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro–in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.
Transfection efficacy of mRNA and DNA using the lipoplex formulation, OH4:DOPE, is screened in vivo. In vivo models are focused upon according to the 3R guidelines, namely chick embryo chorioallantoic membrane and zebra fish (Danio rerio) embryo. Pilot studies in mice are performed to allow correlations.
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