Research

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19 pages, 2283 KiB

Open AccessArticle

Efficient Non-Viral Gene Modification of Mesenchymal Stromal Cells from Umbilical Cord Wharton’s Jelly with Polyethylenimine

by Ana Isabel Ramos-Murillo, Elizabeth Rodríguez, Karl Beltrán, Cristian Ricaurte, Bernardo Camacho, Gustavo Salguero and Rubén Darío Godoy-Silva

Pharmaceutics 2020, 12(9), 896; https://doi.org/10.3390/pharmaceutics12090896 - 22 Sep 2020

Cited by 8 | Viewed by 3702

Abstract

Mesenchymal stromal cells (MSC) derived from human umbilical cord Wharton’s jelly (WJ) have a wide therapeutic potential in cell therapy and tissue engineering because of their multipotential capacity, which can be reinforced through gene therapy in order to modulate specific responses. However, reported [...] Read more.

Mesenchymal stromal cells (MSC) derived from human umbilical cord Wharton’s jelly (WJ) have a wide therapeutic potential in cell therapy and tissue engineering because of their multipotential capacity, which can be reinforced through gene therapy in order to modulate specific responses. However, reported methodologies to transfect WJ-MSC using cationic polymers are scarce. Here, WJ-MSC were transfected using 25 kDa branched- polyethylenimine (PEI) and a DNA plasmid encoding GFP. PEI/plasmid complexes were characterized to establish the best transfection efficiencies with lowest toxicity. Expression of MSC-related cell surface markers was evaluated. Likewise, immunomodulatory activity and multipotential capacity of transfected WJ-MSC were assessed by CD2/CD3/CD28-activated peripheral blood mononuclear cells (PBMC) cocultures and osteogenic and adipogenic differentiation assays, respectively. An association between cell number, PEI and DNA content, and transfection efficiency was observed. The highest transfection efficiency (15.3 ± 8.6%) at the lowest toxicity was achieved using 2 ng/μL DNA and 3.6 ng/μL PEI with 45,000 WJ-MSC in a 24-well plate format (200 μL). Under these conditions, there was no significant difference between the expression of MSC-identity markers, inhibitory effect on CD3+ T lymphocytes proliferation and osteogenic/adipogenic differentiation ability of transfected WJ-MSC, as compared with non-transfected cells. These results suggest that the functional properties of WJ-MSC were not altered after optimized transfection with PEI. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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23 pages, 7454 KiB

Open AccessArticle

Development of Theranostic Cationic Liposomes Designed for Image-Guided Delivery of Nucleic Acid

by Hai Doan Do, Christine Ménager, Aude Michel, Johanne Seguin, Tawba Korichi, Hélène Dhotel, Corinne Marie, Bich-Thuy Doan and Nathalie Mignet

Pharmaceutics 2020, 12(9), 854; https://doi.org/10.3390/pharmaceutics12090854 - 08 Sep 2020

Cited by 11 | Viewed by 3283

Abstract

Cationic liposomes have been considered as potential vectors for gene delivery thanks to their ability to transfect cells with high efficiency. Recently, the combination of diagnostic agent and therapeutic agents in the same particle to form a theranostic system has been reported. Magnetic [...] Read more.

Cationic liposomes have been considered as potential vectors for gene delivery thanks to their ability to transfect cells with high efficiency. Recently, the combination of diagnostic agent and therapeutic agents in the same particle to form a theranostic system has been reported. Magnetic liposomes are one of these examples. Due to the magnetic nanoparticles encapsulated in the liposomes, they can act as a drug delivery system and, at the same time, a magnetic resonance imaging contrast enhancement agent or hyperthermia. In this work, nucleic acid delivery systems based on magnetic cationic liposomes (MCLs) were developed. Two different techniques, reverse phase evaporation and cosolvent sonication, were employed for liposome preparation. Both strategies produced MCLs of less than 200 nm with highly positive charge. Enhancement of their transverse and longitudinal relaxivities r₂ and r₁ was obtained with both kinds of magnetic liposomes compared to free magnetic nanoparticles. Moreover, these MCLs showed high capacity to form complexes and transfect CT-26 cells using the antibiotic-free pFAR4-luc plasmid. The transfection enhancement with magnetofection was also carried out in CT26 cells. These results suggested that our MCLs could be a promising candidate for image-guided gene therapy. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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22 pages, 3253 KiB

Open AccessArticle

Protein Expression Knockdown in Cancer Cells Induced by a Gemini Cationic Lipid Nanovector with Histidine-Based Polar Heads

by Natalia Sánchez-Arribas, María Martínez-Negro, Eva M. Villar, Lourdes Pérez, José Osío Barcina, Emilio Aicart, Pablo Taboada, Andrés Guerrero-Martínez and Elena Junquera

Pharmaceutics 2020, 12(9), 791; https://doi.org/10.3390/pharmaceutics12090791 - 21 Aug 2020

Cited by 8 | Viewed by 2889

Abstract

A histidine-based gemini cationic lipid, which had already demonstrated its efficiency as a plasmid DNA (pDNA) nanocarrier, has been used in this work to transfect a small interfering RNA (siRNA) into cancer cells. In combination with the helper lipid monoolein glycerol (MOG), the [...] Read more.

A histidine-based gemini cationic lipid, which had already demonstrated its efficiency as a plasmid DNA (pDNA) nanocarrier, has been used in this work to transfect a small interfering RNA (siRNA) into cancer cells. In combination with the helper lipid monoolein glycerol (MOG), the cationic lipid was used as an antiGFP-siRNA nanovector in a multidisciplinary study. Initially, a biophysical characterization by zeta potential (ζ) and agarose gel electrophoresis experiments was performed to determine the lipid effective charge and confirm siRNA compaction. The lipoplexes formed were arranged in L_α lamellar lyotropic liquid crystal phases with a cluster-type morphology, as cryo-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS) studies revealed. Additionally, in vitro experiments confirmed the high gene knockdown efficiency of the lipid-based nanovehicle as detected by flow cytometry (FC) and epifluorescence microscopy, even better than that of Lipofectamine2000*, the transfecting reagent commonly used as a positive control. Cytotoxicity assays indicated that the nanovector is non-toxic to cells. Finally, using nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS), apolipoprotein A-I and A-II followed by serum albumin were identified as the proteins with higher affinity for the surface of the lipoplexes. This fact could be beyond the remarkable silencing activity of the histidine-based lipid nanocarrier herein presented. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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9 pages, 4387 KiB

Open AccessArticle

Adeno-Associated Virus Serotype 8-Mediated Genetic Labeling of Cholangiocytes in the Neonatal Murine Liver

by Sanghoon Lee, Ping Zhou, Senyo Whyte and Soona Shin

Pharmaceutics 2020, 12(4), 351; https://doi.org/10.3390/pharmaceutics12040351 - 13 Apr 2020

Cited by 8 | Viewed by 2661

Abstract

Determination of the cellular tropism of viral vectors is imperative for designing precise gene therapy. It has been widely accepted that transduction of hepatocytes using adeno-associated virus serotype 8 (AAV8) is a promising approach to correct inborn errors in neonates, but the type [...] Read more.

Determination of the cellular tropism of viral vectors is imperative for designing precise gene therapy. It has been widely accepted that transduction of hepatocytes using adeno-associated virus serotype 8 (AAV8) is a promising approach to correct inborn errors in neonates, but the type of neonatal hepatic cells transduced by AAV8 has not been thoroughly investigated. To address this question, we used a reporter mouse that carries Cre recombinase (Cre)-inducible yellow fluorescent protein (YFP). Our analysis primarily focused on cholangiocytes, given their pivotal roles in normal liver function and disease. We treated Rosa^YFP/+ mice at postnatal day 2 (P2) with AAV8-cytomegalovirus (CMV) promoter-Cre and analyzed livers at P10 and P56. The vast majority of HNF4α+ hepatocytes were labeled with YFP at both time points, and 11.6% and 24.4% of CK19+ cholangiocytes were marked at P10 and P56, respectively. We also detected YFP+ cells devoid of hepatocyte and cholangiocyte markers, and a subset of these cells expressed the endothelial and fibroblast marker CD34. Next, we used the hepatocyte-specific thyroxine-binding globulin (TBG) promoter. Surprisingly, AAV8-TBG-Cre marked 6.8% and 30.9% of cholangiocytes at P10 and P56, respectively. These results suggest that AAV8 can be a useful tool for targeting cholangiocytes in neonatal livers. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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16 pages, 3369 KiB

Open AccessArticle

Effects of Tissue Pressure on Transgene Expression Characteristics via Renal Local Administration Routes from Ureter or Renal Artery in the Rat Kidney

by Natsuko Oyama, Haruyuki Takahashi, Maho Kawaguchi, Hirotaka Miyamoto, Koyo Nishida, Masako Tsurumaru, Mikiro Nakashima, Fumiyoshi Yamashita, Mitsuru Hashida and Shigeru Kawakami

Pharmaceutics 2020, 12(2), 114; https://doi.org/10.3390/pharmaceutics12020114 - 01 Feb 2020

Cited by 7 | Viewed by 3351

Abstract

We previously developed a renal pressure-mediated transfection method (renal pressure method) as a kidney-specific in vivo gene delivery system. However, additional information on selecting other injection routes and applicable animals remains unclear. In this study, we selected renal arterial and ureteral injections as [...] Read more.

We previously developed a renal pressure-mediated transfection method (renal pressure method) as a kidney-specific in vivo gene delivery system. However, additional information on selecting other injection routes and applicable animals remains unclear. In this study, we selected renal arterial and ureteral injections as local administration routes and evaluated the characteristics of gene delivery such as efficacy, safety, and distribution in pressured kidney of rat. Immediately after the naked pDNA injection, via renal artery or ureter, the left kidney of the rat was pressured using a pressure controlling device. Transfection efficiency of the pressured kidney was about 100-fold higher than that of the injection only group in both administration routes. The optimal pressure intensity in the rat kidney was 1.2 N/cm² for renal arterial injection and 0.9 N/cm² for ureteral injection. We found that transgene expression site differs according to administration route: cortical fibroblasts and renal tubule in renal arterial injection and cortical and medullary tubule and medullary collecting duct in ureteral injection. This is the first report to demonstrate that the renal pressure method can also be effective, after renal arterial and ureteral injections, in rat kidney. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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13 pages, 1835 KiB

Open AccessArticle

Effect of Protein Corona on The Transfection Efficiency of Lipid-Coated Graphene Oxide-Based Cell Transfection Reagents

by Erica Quagliarini, Riccardo Di Santo, Sara Palchetti, Gianmarco Ferri, Francesco Cardarelli, Daniela Pozzi and Giulio Caracciolo

Pharmaceutics 2020, 12(2), 113; https://doi.org/10.3390/pharmaceutics12020113 - 30 Jan 2020

Cited by 16 | Viewed by 3831

Abstract

Coating graphene oxide nanoflakes with cationic lipids leads to highly homogeneous nanoparticles (GOCL NPs) with optimised physicochemical properties for gene delivery applications. In view of in vivo applications, here we use dynamic light scattering, micro-electrophoresis and one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis [...] Read more.

Coating graphene oxide nanoflakes with cationic lipids leads to highly homogeneous nanoparticles (GOCL NPs) with optimised physicochemical properties for gene delivery applications. In view of in vivo applications, here we use dynamic light scattering, micro-electrophoresis and one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis to explore the bionano interactions between GOCL/DNA complexes (hereafter referred to as ”grapholipoplexes”) and human plasma. When exposed to increasing protein concentrations, grapholipoplexes get covered by a protein corona that evolves with protein concentration, leading to biocoronated complexes with modified physicochemical properties. Here, we show that the formation of a protein corona dramatically changes the interactions of grapholipoplexes with four cancer cell lines: two breast cancer cell lines (MDA-MB and MCF-7 cells), a malignant glioma cell line (U-87 MG) and an epithelial colorectal adenocarcinoma cell line (CACO-2). Luciferase assay clearly indicates a monotonous reduction of the transfection efficiency of biocoronated grapholipoplexes as a function of protein concentration. Finally, we report evidence that a protein corona formed at high protein concentrations (as those present in in vivo studies) promotes a higher capture of biocoronated grapholipoplexes within degradative intracellular compartments (e.g., lysosomes), with respect to their pristine counterparts. On the other hand, coronas formed at low protein concentrations (human plasma = 2.5%) lead to high transfection efficiency with no appreciable cytotoxicity. We conclude with a critical assessment of relevant perspectives for the development of novel biocoronated gene delivery systems. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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20 pages, 3552 KiB

Open AccessArticle

Combined Modality Therapy Based on Hybrid Gold Nanostars Coated with Temperature Sensitive Liposomes to Overcome Paclitaxel-Resistance in Hepatic Carcinoma

by Hongyan Zhu, Weili Han, Ye Gan, Qiaofeng Li, Xiaolan Li, Lanlan Shao, Dan Zhu and Hongwei Guo

Pharmaceutics 2019, 11(12), 683; https://doi.org/10.3390/pharmaceutics11120683 - 15 Dec 2019

Cited by 15 | Viewed by 2980

Abstract

In this study, we prepared gold nanostar (GNS) composite nanoparticles containing siRNA of cyclooxygenase-2(siCOX-2) that were modified by tumor targeting ligand 2-deoxyglucose (DG) and transmembrane peptide 9-poly-D-arginine (9R) to form siCOX-2(9R/DG-GNS). Paclitaxel loaded temperature sensitive liposomes (PTX-TSL) were surface-modified to produce [...] Read more.

In this study, we prepared gold nanostar (GNS) composite nanoparticles containing siRNA of cyclooxygenase-2(siCOX-2) that were modified by tumor targeting ligand 2-deoxyglucose (DG) and transmembrane peptide 9-poly-D-arginine (9R) to form siCOX-2(9R/DG-GNS). Paclitaxel loaded temperature sensitive liposomes (PTX-TSL) were surface-modified to produce PTX-TSL-siCOX-2(9R/DG-GNS) displaying homogeneous star-shaped structures of suitable size (293.93 nm ± 3.21) and zeta potentials (2.47 mV ± 0.22). PTX-TSL-siCOX-2(9R/DG-GNS) had a high thermal conversion efficiency under 808 nm laser radiation and a superior transfection efficiency, which may be related to the targeting effects of DG and increased heat induced membrane permeability. COX-2 expression in HepG2/PTX cells was significantly suppressed by PTX-TSL-siCOX-2(9R/DG-GNS) in high temperatures. The co-delivery system inhibited drug-resistant cell growth rates by ≥77% and increased the cell apoptosis rate about 47% at elevated temperatures. PTX-TSL and siCOX-2 loaded gold nanostar particles, therefore, show promise for overcoming tumor resistance. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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Review

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20 pages, 2517 KiB

Open AccessReview

Large-Scale Production of Lentiviral Vectors: Current Perspectives and Challenges

by Eduardo Martínez-Molina, Carlos Chocarro-Wrona, Daniel Martínez-Moreno, Juan A. Marchal and Houria Boulaiz

Pharmaceutics 2020, 12(11), 1051; https://doi.org/10.3390/pharmaceutics12111051 - 03 Nov 2020

Cited by 38 | Viewed by 10276

Abstract

Lentiviral vectors (LVs) have gained value over recent years as gene carriers in gene therapy. These viral vectors are safer than what was previously being used for gene transfer and are capable of infecting both dividing and nondividing cells with a long-term expression. [...] Read more.

Lentiviral vectors (LVs) have gained value over recent years as gene carriers in gene therapy. These viral vectors are safer than what was previously being used for gene transfer and are capable of infecting both dividing and nondividing cells with a long-term expression. This characteristic makes LVs ideal for clinical research, as has been demonstrated with the approval of lentivirus-based gene therapies from the Food and Drug Administration and the European Agency for Medicine. A large number of functional lentiviral particles are required for clinical trials, and large-scale production has been challenging. Therefore, efforts are focused on solving the drawbacks associated with the production and purification of LVsunder current good manufacturing practice. In recent years, we have witnessed the development and optimization of new protocols, packaging cell lines, and culture devices that are very close to reaching the target production level. Here, we review the most recent, efficient, and promising methods for the clinical-scale production ofLVs. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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14 pages, 1378 KiB

Open AccessReview

Microbubbles and Nanobubbles with Ultrasound for Systemic Gene Delivery

by Yoko Endo-Takahashi and Yoichi Negishi

Pharmaceutics 2020, 12(10), 964; https://doi.org/10.3390/pharmaceutics12100964 - 14 Oct 2020

Cited by 57 | Viewed by 4417

Abstract

The regulation of gene expression is a promising therapeutic approach for many intractable diseases. However, its use in clinical applications requires the efficient delivery of nucleic acids to target tissues, which is a major challenge. Recently, various delivery systems employing physical energy, such [...] Read more.

The regulation of gene expression is a promising therapeutic approach for many intractable diseases. However, its use in clinical applications requires the efficient delivery of nucleic acids to target tissues, which is a major challenge. Recently, various delivery systems employing physical energy, such as ultrasound, magnetic force, electric force, and light, have been developed. Ultrasound-mediated delivery has particularly attracted interest due to its safety and low costs. Its delivery effects are also enhanced when combined with microbubbles or nanobubbles that entrap an ultrasound contrast gas. Furthermore, ultrasound-mediated nucleic acid delivery could be performed only in ultrasound exposed areas. In this review, we summarize the ultrasound-mediated nucleic acid systemic delivery system, using microbubbles or nanobubbles, and discuss its possibilities as a therapeutic tool. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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31 pages, 3089 KiB

Open AccessReview

Non-Viral Targeted Nucleic Acid Delivery: Apply Sequences for Optimization

by Yanfang Wang and Ernst Wagner

Pharmaceutics 2020, 12(9), 888; https://doi.org/10.3390/pharmaceutics12090888 - 18 Sep 2020

Cited by 13 | Viewed by 3714

Abstract

In nature, genomes have been optimized by the evolution of their nucleic acid sequences. The design of peptide-like carriers as synthetic sequences provides a strategy for optimizing multifunctional targeted nucleic acid delivery in an iterative process. The optimization of sequence-defined nanocarriers differs for [...] Read more.

In nature, genomes have been optimized by the evolution of their nucleic acid sequences. The design of peptide-like carriers as synthetic sequences provides a strategy for optimizing multifunctional targeted nucleic acid delivery in an iterative process. The optimization of sequence-defined nanocarriers differs for different nucleic acid cargos as well as their specific applications. Supramolecular self-assembly enriched the development of a virus-inspired non-viral nucleic acid delivery system. Incorporation of DNA barcodes presents a complementary approach of applying sequences for nanocarrier optimization. This strategy may greatly help to identify nucleic acid carriers that can overcome pharmacological barriers and facilitate targeted delivery in vivo. Barcode sequences enable simultaneous evaluation of multiple nucleic acid nanocarriers in a single test organism for in vivo biodistribution as well as in vivo bioactivity. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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31 pages, 2559 KiB

Open AccessReview

The Multifaceted Histidine-Based Carriers for Nucleic Acid Delivery: Advances and Challenges

by Jiaxi He, Songhui Xu and A. James Mixson

Pharmaceutics 2020, 12(8), 774; https://doi.org/10.3390/pharmaceutics12080774 - 14 Aug 2020

Cited by 28 | Viewed by 4146

Abstract

Histidines incorporated into carriers of nucleic acids may enhance the extracellular stability of the nanoparticle, yet aid in the intracellular disruption of the nanoparticle, enabling the release of the nucleic acid. Moreover, protonation of histidines in the endosomes may result in endosomal swelling [...] Read more.

Histidines incorporated into carriers of nucleic acids may enhance the extracellular stability of the nanoparticle, yet aid in the intracellular disruption of the nanoparticle, enabling the release of the nucleic acid. Moreover, protonation of histidines in the endosomes may result in endosomal swelling with subsequent lysis. These properties of histidine are based on its five-member imidazole ring in which the two nitrogen atoms may form hydrogen bonds or act as a base in acidic environments. A wide variety of carriers have integrated histidines or histidine-rich domains, which include peptides, polyethylenimine, polysaccharides, platform delivery systems, viral phages, mesoporous silica particles, and liposomes. Histidine-rich carriers have played key roles in our understanding of the stability of nanocarriers and the escape of the nucleic acids from endosomes. These carriers show great promise and offer marked potential in delivering plasmids, siRNA, and mRNA to their intracellular targets. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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29 pages, 3785 KiB

Open AccessReview

Niosome-Based Approach for In Situ Gene Delivery to Retina and Brain Cortex as Immune-Privileged Tissues

by Nuseibah AL Qtaish, Idoia Gallego, Ilia Villate-Beitia, Myriam Sainz-Ramos, Tania Belén López-Méndez, Santiago Grijalvo, Ramón Eritja, Cristina Soto-Sánchez, Gema Martínez-Navarrete, Eduardo Fernández, Gustavo Puras and José Luis Pedraz

Pharmaceutics 2020, 12(3), 198; https://doi.org/10.3390/pharmaceutics12030198 - 25 Feb 2020

Cited by 32 | Viewed by 4080

Abstract

Non-viral vectors have emerged as a promising alternative to viral gene delivery systems due to their safer profile. Among non-viral vectors, recently, niosomes have shown favorable properties for gene delivery, including low toxicity, high stability, and easy production. The three main components of [...] Read more.

Non-viral vectors have emerged as a promising alternative to viral gene delivery systems due to their safer profile. Among non-viral vectors, recently, niosomes have shown favorable properties for gene delivery, including low toxicity, high stability, and easy production. The three main components of niosome formulations include a cationic lipid that is responsible for the electrostatic interactions with the negatively charged genetic material, a non-ionic surfactant that enhances the long-term stability of the niosome, and a helper component that can be added to improve its physicochemical properties and biological performance. This review is aimed at providing recent information about niosome-based non-viral vectors for gene delivery purposes. Specially, we will discuss the composition, preparation methods, physicochemical properties, and biological evaluation of niosomes and corresponding nioplexes that result from the addition of the genetic material onto their cationic surface. Next, we will focus on the in situ application of such niosomes to deliver the genetic material into immune-privileged tissues such as the brain cortex and the retina. Finally, as future perspectives, non-invasive administration routes and different targeting strategies will be discussed. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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23 pages, 1915 KiB

Open AccessReview

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

by Nina Bono, Federica Ponti, Diego Mantovani and Gabriele Candiani

Pharmaceutics 2020, 12(2), 183; https://doi.org/10.3390/pharmaceutics12020183 - 21 Feb 2020

Cited by 104 | Viewed by 8228

Abstract

Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in [...] Read more.

Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. This review provides a catalog of conditions optimized for the gold standard and internal reference, 25 kDa polyethyleneimine, that can be profitably replicated across studies for the sake of comparison. Overall, we wish to pave the way for the implementation of standardized protocols in order to make the evaluation of the effectiveness of transfectants as unbiased as possible. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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16 pages, 758 KiB

Open AccessReview

Production and Application of Multicistronic Constructs for Various Human Disease Therapies

by Alisa A. Shaimardanova, Daria S. Chulpanova, Kristina V. Kitaeva, Ilmira I. Abdrakhmanova, Vladislav M. Chernov, Catrin S. Rutland, Albert A. Rizvanov and Valeriya V. Solovyeva

Pharmaceutics 2019, 11(11), 580; https://doi.org/10.3390/pharmaceutics11110580 - 06 Nov 2019

Cited by 26 | Viewed by 8861

Abstract

The development of multicistronic vectors has opened up new opportunities to address the fundamental issues of molecular and cellular biology related to the need for the simultaneous delivery and joint expression of several genes. To date, the examples of the successful use of [...] Read more.

The development of multicistronic vectors has opened up new opportunities to address the fundamental issues of molecular and cellular biology related to the need for the simultaneous delivery and joint expression of several genes. To date, the examples of the successful use of multicistronic vectors have been described for the development of new methods of treatment of various human diseases, including cardiovascular, oncological, metabolic, autoimmune, and neurodegenerative disorders. The safety and effectiveness of the joint delivery of therapeutic genes in multicistronic vectors based on the internal ribosome entry site (IRES) and self-cleaving 2A peptides have been shown in both in vitro and in vivo experiments as well as in clinical trials. Co-expression of several genes in one vector has also been used to create animal models of various inherited diseases which are caused by mutations in several genes. Multicistronic vectors provide expression of all mutant genes, which allows the most complete mimicking disease pathogenesis. This review comprehensively discusses multicistronic vectors based on IRES nucleotide sequence and self-cleaving 2A peptides, including its features and possible application for the treatment and modeling of various human diseases. Full article

(This article belongs to the Special Issue Gene Delivery Vectors and Physical Methods: Present and Future Trends)

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