scholarly journals Baculovirus-Mediated Gene Delivery into Mammalian Cells Does Not Alter Their Transcriptional and Differentiating Potential but Is Accompanied by Early Viral Gene Expression

2006 ◽  
Vol 80 (8) ◽  
pp. 4135-4146 ◽  
Author(s):  
Christos Kenoutis ◽  
Rodica C. Efrose ◽  
Luc Swevers ◽  
Alexandros A. Lavdas ◽  
Maria Gaitanou ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Schwann Cells ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Primary Cultures ◽  
Recombinant Baculovirus ◽  
Hek293 Cells ◽  
Viral Gene ◽  
Transcriptional Responses ◽  
Baculovirus Infection

ABSTRACT Gene delivery to neural cells is central to the development of transplantation therapies for neurological diseases. In this study, we used a baculovirus derived from the domesticated silk moth, Bombyx mori, as vector for transducing a human cell line (HEK293) and primary cultures of rat Schwann cells. Under optimal conditions of infection with a recombinant baculovirus containing the reporter green fluorescent protein gene under mammalian promoter control, the infected cells express the transgene with high efficiency. Toxicity assays and transcriptome analyses suggest that baculovirus infection is not cytotoxic and does not induce differential transcriptional responses in HEK293 cells. Infected Schwann cells retain their characteristic morphological and molecular phenotype as determined by immunocytochemistry for the marker proteins S-100, glial fibrillary acidic protein, and p75 nerve growth factor receptor. Moreover, baculovirus-infected Schwann cells are capable of differentiating in vitro and express the P0 myelination marker. However, transcripts for several immediate-early viral genes also accumulate in readily detectable levels in the transduced cells. This transcriptional activity raises concerns regarding the long-term safety of baculovirus vectors for gene therapy applications. Potential approaches for overcoming the identified problem are discussed.

scholarly journals Unraveling the Genome-Wide Impact of Recombinant Baculovirus Infection in Mammalian Cells for Gene Delivery

Genes ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1306
Author(s):  
Ha Youn Shin ◽  
Hanul Choi ◽  
Nahyun Kim ◽  
Nayoung Park ◽  
Heesun Kim ◽  
...  
Keyword(s):  
Immune Response ◽  
Gene Delivery ◽  
Mammalian Cells ◽  
Vaccine Development ◽  
Recombinant Baculovirus ◽  
Cytokine Signaling ◽  
Rna Seq ◽  
Immune Response Genes ◽  
Genome Wide ◽  
Baculovirus Infection

Baculovirus expression systems have been widely used to produce recombinant mammalian proteins owing to the lack of viral replication in vertebrates. Although several lines of evidence have demonstrated impacts of baculovirus infection in mammalian hosts, genome-wide effects have not been fully elucidated. Here, we provide comparative transcriptome profiles of baculovirus and host-immune response genes in recombinant baculovirus-infected mammalian and insect cells. Specifically, to decipher the impacts of baculovirus infection in mammalian cells, we conducted total RNA-seq on human 293TT cells and insect Sf9 cells infected with recombinant baculovirus. We found that baculovirus genes were rarely expressed under the control of baculoviral promoters in 293TT cells. Although some baculovirus early genes, such as PE38 and IE-01, showed limited expression in 293TT cells, baculoviral late genes were mostly silent. We also found modest induction of a small number of mammalian immune response genes associated with Toll-like receptors, cytokine signaling, and complement in baculovirus-infected 293TT cells. These comprehensive transcriptome data will contribute to improving recombinant baculovirus as tools for gene delivery, gene therapy, and vaccine development.

scholarly journals Baculovirus as a Tool for Gene Delivery and Gene Therapy

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 510 ◽  
Author(s):  
Chikako Ono ◽  
Toru Okamoto ◽  
Takayuki Abe ◽  
Yoshiharu Matsuura
Keyword(s):  
Gene Delivery ◽  
Mammalian Cells ◽  
Recombinant Baculovirus ◽  
Foreign Gene ◽  
Type I ◽  
Adjuvant Activity ◽  
Baculovirus Infection ◽  
Immunodeficiency Virus ◽  
Host Innate Immune Response ◽  
Infected Cells

Based on its ability to express high levels of protein, baculovirus has been widely used for recombinant protein production in insect cells for more than thirty years with continued technical improvements. In addition, baculovirus has been successfully applied for foreign gene delivery into mammalian cells without any viral replication. However, several CpG motifs are present throughout baculoviral DNA and induce an antiviral response in mammalian cells, resulting in the production of pro-inflammatory cytokines and type I interferon through a Toll-like receptor (TLR)-dependent or -independent signaling pathway, and ultimately limiting the efficiency of transgene expression. On the other hand, by taking advantage of this strong adjuvant activity, recombinant baculoviruses encoding neutralization epitopes can elicit protective immunity in mice. Moreover, immunodeficient cells, such as hepatitis C virus (HCV)- or human immunodeficiency virus (HIV)-infected cells, are more susceptible to baculovirus infection than normal cells and are selectively eliminated by the apoptosis-inducible recombinant baculovirus. Here, we summarize the application of baculovirus as a gene expression vector and the mechanism of the host innate immune response induced by baculovirus in mammalian cells. We also discuss the future prospects of baculovirus vectors.

Recent advances in the analysis full/empty capsid ratio and genome integrity of adeno-associated virus (AAV) gene delivery vectors

2020 ◽  
Vol 20 ◽  
Author(s):  
L. Hajba ◽  
A. Guttman
Keyword(s):  
Gene Delivery ◽  
High Efficiency ◽  
Analytical Techniques ◽  
Viral Gene ◽  
Adeno Associated Virus ◽  
Gene Delivery Vectors ◽  
Empty Capsid ◽  
Purification Methods ◽  
Viral Gene Delivery

: Adeno-associated virus (AAV) is one of the most promising viral gene delivery vectors with long-term gene expression and disease correction featuring high efficiency and excellent safety in human clinical trials. During the production of AAV vectors,there are several quality control (QC)parameters that should be rigorously monitored to comply with clini-cal safety and efficacy. This review gives a short summary of the most frequently used AVV production and purification methods,focusing on the analytical techniques applied to determine the full/empty capsid ratio and the integrity of the encapsidated therapeutic DNA of the products.

scholarly journals Long-Circulating Polymeric Nanovectors for Tumor-Selective Gene Delivery

2005 ◽  
Vol 4 (6) ◽  
pp. 615-625 ◽  
Author(s):  
Sushma Kommareddy ◽  
Sandip B. Tiwari ◽  
Mansoor M. Amiji
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Viral Vectors ◽  
High Efficiency ◽  
Transfection Efficiency ◽  
The United States ◽  
Medical Intervention ◽  
Hydrophilic Polymers ◽  
Circulation Time ◽  
Viral Gene

Significant advances in the understanding of the genetic abnormalities that lead to the development, progression, and metastasis of neoplastic diseases has raised the promise of gene therapy as an approach to medical intervention. Most of the clinical protocols that have been approved in the United States for gene therapy have used the viral vectors because of the high efficiency of gene transfer. Conventional means of gene delivery using viral vectors, however, has undesirable side effects such as insertion of mutational viral gene into the host genome and development of replication competent viruses. Among non-viral gene delivery methods, polymeric nanoparticles are increasingly becoming popular as vectors of choice. The major limitation of these nanoparticles is poor transfection efficiency at the target site after systemic administration due to uptake by the cells of reticuloendothelial system (RES). In order to reduce the uptake by the cells of the RES and improve blood circulation time, these nanoparticles are coated with hydrophilic polymers such as poly(ethylene glycol) (PEG). This article reviews the use of such hydrophilic polymers employed for improving the circulation time of the nanocarriers. The mechanism of polymer coating and factors affecting the circulation time of these nanocarriers will be discussed. In addition to the long circulating property, modifications to improve the target specificity of the particles and the limitations of steric protection will be analyzed.

Understanding why the same gene delivery vector behaves differently in different cell types is essential for developing more adaptable transfection systems

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Gene Delivery ◽  
High Efficiency ◽  
Transfection Efficiency ◽  
Cell Metabolism ◽  
New Technology ◽  
Cell Types ◽  
Viral Gene ◽  
Proliferation Capacity ◽  
Delivery Vector ◽  
Different Cell Types

Since their origin, non-viral gene delivery reagents have evolved into a variety of effective delivery reagents with a variety of components and designs, and are widely used in gene therapy and gene engineering. A flood of successful commercial gene delivery reagents has also developed, and PEI has emerged as the "gold standard" for the industry. On the other hand, their transfection efficiency must be enhanced and their cell toxicity must be reduced. In recent years, toxicity, efficiency and targeted investigations have progressed. In addition to creating and manufacturing reagents with reduced toxicity and higher efficiency, polypeptides that stimulate cell membrane perforation and tiny molecular compounds that can better compress pDNA, as well as various combinations with liposomes or polymer vectors, have demonstrated improved outcomes. However, most of these freshly created delivery vector reagents are still under investigation, and others require additional refinement to achieve high transfection efficiency and minimum toxicity. The processes behind the effects of various gene delivery reagents, genes, and drugs entering cells, as well as their transit, escape, and cell metabolism, are also unclear. This requires improving relevant research. Understanding why the same reagent reacts differently to different cell types is crucial to creating more adaptive transfection reagents for different cell lines. This is suggested because different cells have different growth cycles. Because of their weak proliferation capacity, primordial cells, for example, are harder to replicate.Artificial intelligence, real-world and virtual-world integration technology, big data, multiomics technology, and signal pathway research have all achieved substantial breakthroughs in recent years, and novel transfection reagents and drug delivery technologies are predicted to continue. It is worth examining how to take advantage of the scientific and high-efficiency benefits that new technology provides for research and how to solve the issues given by the in-depth examination of the selection and mechanism of action of novel composite materials in vector reagent creation.

scholarly journals Enhanced transfection of a macromolecular lignin-based DNA complex with low cellular toxicity

2018 ◽  
Vol 38 (6) ◽  
Author(s):  
Yoon Khei Ho ◽  
Dan Kai ◽  
Geraldine Xue En Tu ◽  
G. Roshan Deen ◽  
Heng Phon Too ◽  
...  
Keyword(s):  
Gene Delivery ◽  
High Efficiency ◽  
Endosomal Escape ◽  
Viral Gene ◽  
Attractive Alternative ◽  
Cationic Polymers ◽  
Dna Amount ◽  
Cellular Toxicity ◽  
Molar Ratios ◽  
Post Transfection

Cationic polymers remain attractive tools for non-viral gene transfer. The effectiveness of these vectors rely on the ability to deliver plasmid DNA (pDNA) into the nucleus of cells. While we have previously demonstrated the potential of Lignin-PGEA-PEGMA as a non-viral gene delivery vector, alterations of cellular phenotype and cytotoxicity were observed post transfection. The present study aims to explore transfection conditions for high efficiency and low toxicity of the Lignin-PGEA-PEGMA based gene delivery system. Cellular toxicity was significantly reduced by using the centrifugation protocol, which enables rapid deposition of DNA complexes. Replacement of media post centrifugation resulted in minimal exposure of cells to excess polymers, which were toxic to cells. At an optimized DNA amount (500–750 ng) and molar ratios of nitrogen (N) in polymer to phosphate (P) in pDNA (N/P = 30–40), with the use of a novel transfection enhancer that facilitates endosomal escape and nuclear trafficking, the efficiency of gene delivery was increased significantly 24 h post transfection. The present study demonstrated an appropriately optimized protocol that enabled the utility of a novel cationic polymer blend with a mixture of fusogenic lipids and a histone deacetylate inhibitor in non-viral transfection, thereby providing an attractive alternative to costly commercial gene carriers.

scholarly journals Single-cell analysis of antiviral neuroinflammatory responses in the mouse dorsal raphe nucleus

2019 ◽  
Author(s):  
Kee Wui Huang ◽  
Bernardo L. Sabatini
Keyword(s):  
Gene Delivery ◽  
Single Cell ◽  
Dorsal Raphe Nucleus ◽  
Single Cell Analysis ◽  
Critical Evaluation ◽  
Dorsal Raphe ◽  
Raphe Nucleus ◽  
Viral Gene ◽  
Transcriptional Responses ◽  
Dorsal Raphé

ABSTRACTNeuroinflammatory processes have been implicated in neurodegenerative and psychiatric diseases, and limit the utility of viruses for gene delivery. Here we analyzed 60,212 single-cell RNA profiles to assess both global and cell type-specific transcriptional responses in the mouse dorsal raphe nucleus following axonal infection of neurons by rabies viruses. We identified several leukocyte populations, which infiltrate the brain, that are distinct from resident immune cells. Additionally, we uncovered transcriptionally distinct states of microglia along an activation trajectory that may serve different functions, ranging from surveillance to antigen presentation and cytokine secretion. Our study also provides a critical evaluation of the compatibility between rabies-mediated connectivity mapping and single-cell transcriptional profiling. These findings provide additional insights into the distinct contributions of various cell types in the antiviral response, and will serve as a resource for the design of strategies to circumvent immune responses to improve the efficacy of viral gene delivery.

scholarly journals Supramolecular Surface Functionalization of Iron Oxide Nanoparticles with α-Cyclodextrin-Based Cationic Star Polymer for Magnetically-Enhanced Gene Delivery

Pharmaceutics ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 1884
Author(s):  
Hanyi Li ◽  
Erwin Peng ◽  
Feng Zhao ◽  
Jun Li ◽  
Junmin Xue
Keyword(s):  
Iron Oxide ◽  
Gene Delivery ◽  
Iron Oxide Nanoparticles ◽  
Mammalian Cells ◽  
Gene Transfection ◽  
Functional Materials ◽  
Water Soluble ◽  
Star Polymer ◽  
Viral Gene ◽  
Oxide Nanoparticles

Supramolecular polymers formed through host–guest complexation have inspired many interesting developments of functional materials for biological and biomedical applications. Here, we report a novel design of a non-viral gene delivery system composed of a cationic star polymer forming supramolecular complexes with the surface oleyl groups of superparamagnetic iron oxide nanoparticles (SPIONs), for magnetically enhanced delivery of DNA into mammalian cells. The cationic star polymer was synthesized by grafting multiple oligoethylenimine (OEI) chains onto an α-cyclodextrin (α-CD) core. The SPIONs were synthesized from iron(III) acetylacetonate and stabilized by hydrophobic oleic acid and oleylamine in hexane, which were characterized in terms of their size, structure, morphology, and magnetic properties. The synthesized magnetic particles were found to be superparamagnetic, making them a suitable ferrofluid for biological applications. In order to change the hydrophobic surface of the SPIONs to a hydrophilic surface with functionalities for plasmid DNA (pDNA) binding and gene delivery, a non-traditional but simple supramolecular surface modification process was used. The α-CD-OEI cationic star polymer was dissolved in water and then mixed with the SPIONs stabilized in hexane. The SPIONs were “pulled” into the water phase through the formation of supramolecular host–guest inclusion complexes between the α-CD unit and the oleyl surface of the SPIONs, while the surface of the SPIONs was changed to OEI cationic polymers. The α-CD-OEI-SPION complex could effectively bind and condense pDNA to form α-CD-OEI-SPION/pDNA polyplex nanoparticles at the size of ca. 200 nm suitable for delivery of genes into cells through endocytosis. The cytotoxicity of the α-CD-OEI-SPION complex was also found to be lower than high-molecular-weight polyethylenimine, which was widely studied previously as a standard non-viral gene vector. When gene transfection was carried out in the presence of an external magnetic field, the α-CD-OEI-SPION/pDNA polyplex nanoparticles greatly increased the gene transfection efficiency by nearly tenfold. Therefore, the study has demonstrated a facile two-in-one method to make the SPIONs water-soluble as well as functionalized for enhanced magnetofection.

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