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17 pages, 3205 KiB

Open AccessArticle

Chitosan Oleate Coated PLGA Nanoparticles as siRNA Drug Delivery System

by Dalila Miele, Xin Xia, Laura Catenacci, Milena Sorrenti, Silvia Rossi, Giuseppina Sandri, Franca Ferrari, John J. Rossi and Maria Cristina Bonferoni

Pharmaceutics 2021, 13(10), 1716; https://doi.org/10.3390/pharmaceutics13101716 - 17 Oct 2021

Cited by 22 | Viewed by 3647

Abstract

Oligonucleotide therapeutics such as miRNAs and siRNAs represent a class of molecules developed to modulate gene expression by interfering with ribonucleic acids (RNAs) and protein synthesis. These molecules are characterized by strong instability and easy degradation due to nuclease enzymes. To avoid these drawbacks and ensure efficient delivery to target cells, viral and non-viral vectors are the two main approaches currently employed. Viral vectors are one of the major vehicles in gene therapy; however, the potent immunogenicity and the insertional mutagenesis is a potential issue for the patient. Non-viral vectors, such as polymeric nanocarriers, provide a safer and more efficient delivery of RNA-interfering molecules. The aim of this work is to employ PLGA core nanoparticles shell-coated with chitosan oleate as siRNA carriers. An siRNA targeted on HIV-1, directed against the viral Tat/Rev transcripts was employed as a model. The ionic interaction between the oligonucleotide’s moieties, negatively charged, and the positive surface charges of the chitosan shell was exploited to associate siRNA and nanoparticles. Non-covalent bonds can protect siRNA from nuclease degradation and guarantee a good cell internalization and a fast release of the siRNA into the cytosolic portion, allowing its easy activation. Full article

(This article belongs to the Special Issue Special Issue in Honor of Professor Carla Caramella)

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12 pages, 286 KiB

Open AccessReview

Implications of the EFSA Scientific Opinion on Site Directed Nucleases 1 and 2 for Risk Assessment of Genome-Edited Plants in the EU

by Nils Rostoks

Agronomy 2021, 11(3), 572; https://doi.org/10.3390/agronomy11030572 - 18 Mar 2021

Cited by 13 | Viewed by 4415

Abstract

Genome editing is a set of techniques for introducing targeted changes in genomes. It may be achieved by enzymes collectively called site-directed nucleases (SDN). Site-specificity of SDNs is provided either by the DNA binding domain of the protein molecule itself or by RNA molecule(s) that direct SDN to a specific site in the genome. In contrast to transgenesis resulting in the insertion of exogenous DNA, genome editing only affects specific endogenous sequences. Therefore, multiple jurisdictions around the world have exempted certain types of genome-edited organisms from national biosafety regulations completely, or on a case-by-case basis. In the EU, however, the ruling of the Court of Justice on the scope of mutagenesis exemption case C-528/16 indicated that the genome-edited organisms are subject to the GMO Directive, but the practical implications for stakeholders wishing to develop and authorize genome-edited products in the EU remain unclear. European Food Safety Authority in response to a request by European Commission has produced a scientific opinion on plants developed by SDN-1, SDN-2, and oligonucleotide-directed mutagenesis (ODM) genome editing techniques. In this review, I will (1) provide a conceptual background on GMO risk assessment in the EU; (2) will introduce the main conclusions of the EFSA opinion, and (3) will outline the potential impact on the risk assessment of genome-edited plants. Full article

(This article belongs to the Special Issue Precision Genome Editing for Plant Breeding)

33 pages, 1204 KiB

Open AccessReview

Genome Editing in Agriculture: Technical and Practical Considerations

by Julia Jansing, Andreas Schiermeyer, Stefan Schillberg, Rainer Fischer and Luisa Bortesi

Int. J. Mol. Sci. 2019, 20(12), 2888; https://doi.org/10.3390/ijms20122888 - 13 Jun 2019

Cited by 45 | Viewed by 11666

Abstract

The advent of precise genome-editing tools has revolutionized the way we create new plant varieties. Three groups of tools are now available, classified according to their mechanism of action: Programmable sequence-specific nucleases, base-editing enzymes, and oligonucleotides. The corresponding techniques not only lead to different outcomes, but also have implications for the public acceptance and regulatory approval of genome-edited plants. Despite the high efficiency and precision of the tools, there are still major bottlenecks in the generation of new and improved varieties, including the efficient delivery of the genome-editing reagents, the selection of desired events, and the regeneration of intact plants. In this review, we evaluate current delivery and regeneration methods, discuss their suitability for important crop species, and consider the practical aspects of applying the different genome-editing techniques in agriculture. Full article

(This article belongs to the Special Issue Genome Editing in Plants)

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11 pages, 770 KiB

Open AccessArticle

Solid Phase Assembly of Fully Protected Trinucleotide Building Blocks for Codon-Based Gene Synthesis

by Ruth Suchsland, Bettina Appel, Matthäus Janczyk and Sabine Müller

Appl. Sci. 2019, 9(11), 2199; https://doi.org/10.3390/app9112199 - 29 May 2019

Cited by 5 | Viewed by 4181

Abstract

The use of pre-formed trinucleotides, representing codons of the 20 canonical amino acids, for oligonucleotide-directed mutagenesis offers the advantage of controlled randomization and generation of “smart libraries”. We here present a method for the preparation of fully protected trinucleotides on solid phase. The key issue of our strategy is the linkage of the starting nucleoside to the solid support via a traceless disulfide linker. Upon trinucleotide assembly, the disulfide bridge is cleaved under reducing conditions, and the fully protected trinucleotide is released with a terminal 3′-OH group. Full article

(This article belongs to the Section Chemical and Molecular Sciences)

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24 pages, 2727 KiB

Open AccessReview

Genome Editing Tools in Plants

by Tapan Kumar Mohanta, Tufail Bashir, Abeer Hashem, Elsayed Fathi Abd_Allah and Hanhong Bae

Genes 2017, 8(12), 399; https://doi.org/10.3390/genes8120399 - 19 Dec 2017

Cited by 82 | Viewed by 11918

Abstract

Genome editing tools have the potential to change the genomic architecture of a genome at precise locations, with desired accuracy. These tools have been efficiently used for trait discovery and for the generation of plants with high crop yields and resistance to biotic and abiotic stresses. Due to complex genomic architecture, it is challenging to edit all of the genes/genomes using a particular genome editing tool. Therefore, to overcome this challenging task, several genome editing tools have been developed to facilitate efficient genome editing. Some of the major genome editing tools used to edit plant genomes are: Homologous recombination (HR), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), pentatricopeptide repeat proteins (PPRs), the CRISPR/Cas9 system, RNA interference (RNAi), cisgenesis, and intragenesis. In addition, site-directed sequence editing and oligonucleotide-directed mutagenesis have the potential to edit the genome at the single-nucleotide level. Recently, adenine base editors (ABEs) have been developed to mutate A-T base pairs to G-C base pairs. ABEs use deoxyadeninedeaminase (TadA) with catalytically impaired Cas9 nickase to mutate A-T base pairs to G-C base pairs. Full article

(This article belongs to the Section Plant Genetics and Genomics)

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7 pages, 183 KiB

Open AccessReview

Biotech Approaches to Overcome the Limitations of Using Transgenic Plants in Organic Farming

by Luca Lombardo and Samanta Zelasco

Sustainability 2016, 8(5), 497; https://doi.org/10.3390/su8050497 - 20 May 2016

Cited by 18 | Viewed by 7526

Abstract

Organic farming prohibits the use of genetically modified organisms (GMOs) inasmuch as their genetic material has been altered in a way that does not occur naturally. In actual fact, there is a conventional identity between GMOs and transgenic organisms, so that genetic modification methods such as somatic hybridization and mutagenesis are equalized to conventional breeding. A loophole in this system is represented by more or less innovative genetic engineering approaches under regulatory discussion, such as cisgenesis, oligonucleotide-directed mutagenesis, and antisense technologies, that are redefining the concept of GMOs and might circumvent the requirements of the GMO legislation and, indirectly, of organic farming. Full article

(This article belongs to the Special Issue Organic Farming and Gene Manipulation)

16 pages, 755 KiB

Open AccessArticle

The DNA-Damage Response to γ-Radiation Is Affected by miR-27a in A549 Cells

by Andrea Di Francesco, Cristiano De Pittà, Francesca Moret, Vito Barbieri, Lucia Celotti and Maddalena Mognato

Int. J. Mol. Sci. 2013, 14(9), 17881-17896; https://doi.org/10.3390/ijms140917881 - 2 Sep 2013

Cited by 33 | Viewed by 8599

Abstract

Perturbations during the cell DNA-Damage Response (DDR) can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs), small non-coding RNAs that act as post-transcriptional regulators of gene expression. The oncogenic miR-27a is over-expressed in several tumors and, in the present study, we investigated its interaction with ATM, the gene coding for the main kinase of DDR pathway. Experimental validation to confirm miR-27a as a direct regulator of ATM was performed by site-direct mutagenesis of the luciferase reporter vector containing the 3'UTR of ATM gene, and by miRNA oligonucleotide mimics. We then explored the functional miR-27a/ATM interaction under biological conditions, i.e., during the response of A549 cells to ionizing radiation (IR) exposure. To evaluate if miR-27a over-expression affects IR-induced DDR activation in A549 cells we determined cell survival, cell cycle progression and DNA double-strand break (DSB) repair. Our results show that up-regulation of miR-27a promotes cell proliferation of non-irradiated and irradiated cells. Moreover, increased expression of endogenous mature miR-27a in A549 cells affects DBS rejoining kinetics early after irradiation. Full article

(This article belongs to the Collection Radiation Toxicity in Cells)

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