Evaluation of the nucleopolyhedrovirus of Anticarsia gemmatalis as a vector for gene therapy in mammals

2020 ◽  
Vol 20 ◽  
Author(s):  
Cintia N. Parsza ◽  
Diego L. Mengual Gómez ◽  
Jorge Alejandro Simonin ◽  
Mariano Nicolás Belaich ◽  
Pablo Daniel Ghiringhelli
Keyword(s):  
Gene Therapy ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Autographa Californica ◽  
Anticarsia Gemmatalis ◽  
Agricultural Pests ◽  
Mammalian Cell Lines ◽  
Delivery Vector ◽  
Wide Range ◽  
Insect Pathogens

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV) has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals, because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. Objective/Method: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. Conclusion: Consequently, this insect pathogen is proposed as an alternative of non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity

1977 ◽  
Vol 23 (2) ◽  
pp. 183-189 ◽  
Author(s):  
John L. Middlebrook ◽  
Rebecca B. Dorland
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Corynebacterium Diphtheriae ◽  
Pseudomonas Exotoxin ◽  
Intact Cells ◽  
Mammalian Cell Lines ◽  
First Order Kinetics ◽  
Wide Range ◽  
Species Specific

The sensitivities of 21 mammalian cell lines to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae were measured. Each line exhibited 1–4 log differences in sensitivities to the two toxins. No species-specific sensitivities were noted for Pseudomonas exotoxin while diphtheria exotoxin was most potent in cells of monkey origin, followed by human and hamster cells. Rat-and mouse-derived cell lines were very in sensitive to diphtheria exotoxin. The rates of cellular intoxication by both toxins exhibited apparent first-order kinetics and were indistinguishable from one another when equipotent doses were used. Our preparation of diphtheria exotoxin appeared to have a slightly higher ADP-ribosylating efficiency than did Pseudomonas toxin. However, neither toxin exhibited cell line–specific differences in ribosylating efficiencies which could have explained the wide range in potencies for intact cells. Our results suggest that there are significant differences in the mechanisms of cellular intoxication by Pseudomonas and diphtheria exotoxins and that these differences probably exist in the attachment or internalization stages of toxin action.

scholarly journals Functional Display of an α2 Integrin-Specific Motif (RKK) on the Surface of Baculovirus Particles

2005 ◽  
Vol 4 (4) ◽  
pp. 437-445 ◽  
Author(s):  
Reetta Riikonen ◽  
Heli Matilainen ◽  
Nina Rajala ◽  
Olli Pentikäinen ◽  
Mark Johnson ◽  
...  
Keyword(s):  
Envelope Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Recombinant Baculovirus ◽  
Autographa Californica ◽  
Wild Type ◽  
Receptor Binding Site ◽  
Mammalian Cell Lines ◽  
Green Fluorescent

The use of baculovirus vectors shows promise as a tool for gene delivery into mammalian cells. These insect viruses have been shown to transduce a variety of mammalian cell lines, and gene transfer has also been demonstrated in vivo. In this study, we generated two recombinant baculovirus vectors displaying an integrin-specific motif, RKK, as a part of two different loops of the green fluorescent protein (GFP) fused with the major envelope protein gp64 of Autographa californica M nucleopolyhedrovirus. By enzyme linked immunosorbent assays, these viruses were shown to bind a peptide representing the receptor binding site of an α2 integrin, the α2I-domain. However, the interaction was not strong enough to overcome binding of wild type gp64 to the unknown cellular receptor(s) on the surface of α2 integrin-expressing cells (CHO-α2β1) or enhance the viral uptake. After treatment of these cells with phospholipase C, internalization of all viruses was blocked or decreased significantly. However, one of the RKK displaying viruses, AcGFP(K)gp64, was still able to internalize into CHO-α2β1 cells, although at a lower level as compared to non-treated cells. This may indicate the possible utilization of a PLC independent alternative route via, in this case, the α2β1 integrin.

scholarly journals Improving promiscuous mammalian cell entry by the baculovirus Autographa californica multiple nuclear polyhedrosis virus

2012 ◽  
Vol 33 (1) ◽  
Author(s):  
Neil M. J. O’Flynn ◽  
Avnish Patel ◽  
Jan Kadlec ◽  
Ian M. Jones
Keyword(s):  
Mammalian Cell ◽  
Mammalian Cells ◽  
Nuclear Polyhedrosis Virus ◽  
Low Ph ◽  
Autographa Californica ◽  
Cell Entry ◽  
Surface Glycoprotein ◽  
Mammalian Cell Lines ◽  
Polyhedrosis Virus ◽  
Nuclear Polyhedrosis

The insect baculovirus AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) enters many mammalian cell lines, prompting its application as a general eukaryotic gene delivery agent, but the basis of entry is poorly understood. For adherent mammalian cells, we show that entry is favoured by low pH and by increasing the available cell-surface area through a transient release from the substratum. Low pH also stimulated baculovirus entry into mammalian cells grown in suspension which, optimally, could reach 90% of the transduced population. The basic loop, residues 268–281, of the viral surface glycoprotein gp64 was required for entry and a tetra mutant with increasing basicity increased entry into a range of mammalian cells. The same mutant failed to plaque in Sf9 cells, instead showing individual cell entry and minimal cell-to-cell spread, consistent with an altered fusion phenotype. Viruses grown in different insect cells showed different mammalian cell entry efficiencies, suggesting that additional factors also govern entry.

The Wilson's disease protein expressed in Sf9 cells is phosphorylated

2002 ◽  
Vol 30 (4) ◽  
pp. 739-741 ◽  
Author(s):  
S. M. Vanderwerf ◽  
S. Lutsenko
Keyword(s):  
Mammalian Cells ◽  
Wilson’S Disease ◽  
Insect Cells ◽  
Physiological Role ◽  
Wilson's Disease ◽  
Copper Transporter ◽  
Mammalian Cell Lines ◽  
Disease Protein ◽  
Baculovirus System

The Wilson's disease protein (WNDP), a copper transporter, is a crucial mediator of copper homoeostasis in mammalian cells. We recently found that changes in copper concentration regulate the phosphorylation level of WNDP. WNDP phosphorylation was observed in several mammalian cell lines, suggesting that a common phosphorylation pathway exists in these cells. Here we demonstrate that WNDP expressed in Sf9 insect cells is also phosphorylated, as evidenced by metabolic labelling of these cells with [32P]Pi. Because the baculovirus system allows us to generate large amounts of protein, we are using this expression method to isolate WNDP and map the sites of WNDP phosphorylation. The identification of phosphorylation sites is the first step towards understanding the physiological role of WNDP phosphorylation.

scholarly journals Ligand-Directed Gene Targeting to Mammalian Cells by Pseudotype Baculoviruses

2005 ◽  
Vol 79 (6) ◽  
pp. 3639-3652 ◽  
Author(s):  
Yoshinori Kitagawa ◽  
Hideki Tani ◽  
Chang Kwang Limn ◽  
Tomoko M. Matsunaga ◽  
Kohji Moriishi ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Reporter Gene ◽  
Mammalian Cells ◽  
Measles Virus ◽  
Insect Cells ◽  
Viral Vector ◽  
Target Cells ◽  
Gene Transduction ◽  
Mammalian Cell Lines ◽  
Recombinant Bacmids

ABSTRACT The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can infect a variety of mammalian cells, as well as insect cells, facilitating its use as a viral vector for gene delivery into mammalian cells. Glycoprotein gp64, a major component of the budded AcMNPV envelope, is involved in viral entry into cells by receptor-mediated endocytosis and subsequent membrane fusion. We examined the potential production of pseudotype baculovirus particles transiently carrying ligands of interest in place of gp64 as a method of ligand-directed gene delivery into target cells. During amplification of a gp64-null pseudotype baculovirus carrying a green fluorescent protein gene in gp64-expressing insect cells, however, we observed the high-frequency appearance of a replication-competent virus incorporating the gp64 gene into the viral genome. To avoid generation of replication-competent revertants, we prepared pseudotype baculoviruses by transfection with recombinant bacmids without further amplification in the gp64-expressing cells. We constructed gp64-null recombinant bacmids carrying cDNAs encoding either vesicular stomatitis virus G protein (VSVG) or measles virus receptors (CD46 or SLAM). The VSVG pseudotype baculovirus efficiently transduced a reporter gene into a variety of mammalian cell lines, while CD46 and SLAM pseudotype baculoviruses allowed ligand-receptor-directed reporter gene transduction into target cells expressing measles virus envelope glycoproteins. Gene transduction mediated by the pseudotype baculoviruses could be inhibited by pretreatment with specific antibodies. These results indicate the possible application of pseudotype baculoviruses in ligand-directed gene delivery into target cells.

scholarly journals Novel Fluorescent Technology Platform for High Throughput Cytotoxicity and Proliferation Assays

2000 ◽  
Vol 5 (3) ◽  
pp. 141-152 ◽  
Author(s):  
Magdalena Wodnicka ◽  
Richard D. Guarino ◽  
John J. Hemperly ◽  
Mark R. Timmins ◽  
David Stitt ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Number ◽  
Assay Method ◽  
Toxic Substances ◽  
Technology Platform ◽  
Biosensor Technology ◽  
Mammalian Cell Lines ◽  
Automation Systems ◽  
Wide Range ◽  
Mtt Assays

We have developed a novel fluorescent Oxygen BioSensor technology platform adaptable to many applications in the area of drug discovery and development, particularly cell-based assays. This biosensor technology requires no additional reagents or incubations, and affords continuous real-time readout of dissolved oxygen concentrations. Since the level of oxygen dissolved in an assay's medium correlates to the number and viability of the cells in the medium, this technology is ideally suited for monitoring cell viability, proliferation, or death. The technology is particularly well suited to investigating cells' kinetic responses to proliferative or toxic stimuli, such as drugs. When incorporated into a 96- or 384-well microplate format, it is compatible with standard laboratory automation systems. Here we present data illustrating the application of the Oxygen BioSensor technology for rapid, homogeneous detection and evaluation of metabolic activity of a variety of eukaryotic and prokaryotic cells, including mammalian cells, insect cells, yeast, and bacteria. In the absence of toxic substances, we find a good correlation between cell number and signal over a wide range of cell concentrations and growth times. To evaluate the usefulness of the Oxygen BioSensor for cytotoxicity assays, we have performed a series of experiments using a range of toxic agents and cell types, including both bacteria and mammalian cell lines. In a side-by-side comparison to standard MTT assays using HL60 cells, comparable IC50 values were found with the Oxygen BioSensor for five different toxins or drugs. This assay method does not have the need for additional reagents, handling steps, or incubation periods required by the MTT assays.

Exosome as a Natural Gene Delivery Vector for Cancer Treatment

2020 ◽  
Vol 20 (11) ◽  
pp. 821-830
Author(s):  
Prasad Pofali ◽  
Adrita Mondal ◽  
Vaishali Londhe
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Nucleic Acids ◽  
Cancer Treatment ◽  
Intestinal Barrier ◽  
Gene Carrier ◽  
Gene Delivery Vector ◽  
Delivery Vector

Background: Current gene therapy vectors such as viral, non-viral, and bacterial vectors, which are used for cancer treatment, but there are certain safety concerns and stability issues of these conventional vectors. Exosomes are the vesicles of size 40-100 nm secreted from multivesicular bodies into the extracellular environment by most of the cell types in-vivo and in-vitro. As a natural nanocarrier, exosomes are immunologically inert, biocompatible, and can cross biological barriers like the blood-brain barrier, intestinal barrier, and placental barrier. Objective: This review focusses on the role of exosome as a carrier to efficiently deliver a gene for cancer treatment and diagnosis. The methods for loading of nucleic acids onto the exosomes, advantages of exosomes as a smart intercellular shuttle for gene delivery and therapeutic applications as a gene delivery vector for siRNA, miRNA and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and also the limitations of exosomes as a gene carrier are all reviewed in this article. Methods: Mostly, electroporation and chemical transfection are used to prepare gene loaded exosomes. Results: Exosome-mediated delivery is highly promising and advantageous in comparison to the current delivery methods for systemic gene therapy. Targeted exosomes, loaded with therapeutic nucleic acids, can efficiently promote the reduction of tumor proliferation without any adverse effects. Conclusion: In the near future, exosomes can become an efficient gene carrier for delivery and a biomarker for the diagnosis and treatment of cancer.

scholarly journals Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 481
Author(s):  
Gemma G. Martínez-García ◽  
Raúl F. Pérez ◽  
Álvaro F. Fernández ◽  
Sylvere Durand ◽  
Guido Kroemer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Mammalian Cells ◽  
Tissue Homeostasis ◽  
In Vivo Studies ◽  
Protective Mechanism ◽  
Cardiac Damage ◽  
Wide Range ◽  
First Time

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

scholarly journals Microneedle Arrays Combined with Nanomedicine Approaches for Transdermal Delivery of Therapeutics

2021 ◽  
Vol 10 (2) ◽  
pp. 181
Author(s):  
Vahid Alimardani ◽  
Samira Sadat Abolmaali ◽  
Gholamhossein Yousefi ◽  
Zahra Rahiminezhad ◽  
Mehdi Abedi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transdermal Delivery ◽  
Transdermal Drug Delivery ◽  
Skin Barrier ◽  
Skin Penetration ◽  
Therapeutic Agents ◽  
Inorganic Nanoparticles ◽  
Microneedle Array ◽  
Wide Range ◽  
Microneedle Arrays

Organic and inorganic nanoparticles (NPs) have shown promising outcomes in transdermal drug delivery. NPs can not only enhance the skin penetration of small/biomacromolecule therapeutic agents but can also impart control over drug release or target impaired tissue. Thanks to their unique optical, photothermal, and superparamagnetic features, NPs have been also utilized for the treatment of skin disorders, imaging, and biosensing applications. Despite the widespread transdermal applications of NPs, their delivery across the stratum corneum, which is the main skin barrier, has remained challenging. Microneedle array (MN) technology has recently revealed promising outcomes in the delivery of various formulations, especially NPs to deliver both hydrophilic and hydrophobic therapeutic agents. The present work reviews the advancements in the application of MNs and NPs for an effective transdermal delivery of a wide range of therapeutics in cancer chemotherapy and immunotherapy, photothermal and photodynamic therapy, peptide/protein vaccination, and the gene therapy of various diseases. In addition, this paper provides an overall insight on MNs’ challenges and summarizes the recent achievements in clinical trials with future outlooks on the transdermal delivery of a wide range of nanomedicines.

scholarly journals Bacmid Expression of Granulovirus Enhancin En3 Accumulates in Cell Soluble Fraction to Potentiate Nucleopolyhedrovirus Infection

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1233
Author(s):  
Adriana Ricarte-Bermejo ◽  
Oihane Simón ◽  
Ana Beatriz Fernández ◽  
Trevor Williams ◽  
Primitivo Caballero
Keyword(s):  
Trichoplusia Ni ◽  
Recombinant Virus ◽  
Spodoptera Exigua ◽  
Insect Cells ◽  
Soluble Fraction ◽  
Autographa Californica ◽  
Insect Midgut ◽  
Baculovirus Infection ◽  
Occlusion Bodies ◽  
Infected Cells

Enhancins are metalloproteinases that facilitate baculovirus infection in the insect midgut. They are more prevalent in granuloviruses (GVs), constituting up to 5% of the proteins of viral occlusion bodies (OBs). In nucleopolyhedroviruses (NPVs), in contrast, they are present in the envelope of the occlusion-derived virions (ODV). In the present study, we constructed a recombinant Autographa californica NPV (AcMNPV) that expressed the Trichoplusia ni GV (TnGV) enhancin 3 (En3), with the aim of increasing the presence of enhancin in the OBs or ODVs. En3 was successfully produced but did not localize to the OBs or the ODVs and accumulated in the soluble fraction of infected cells. As a result, increased OB pathogenicity was observed when OBs were administered in mixtures with the soluble fraction of infected cells. The mixture of OBs and the soluble fraction of Sf9 cells infected with BacPhEn3 recombinant virus was ~3- and ~4.7-fold more pathogenic than BacPh control OBs in the second and fourth instars of Spodoptera exigua, respectively. In contrast, when purified, recombinant BacPhEn3 OBs were as pathogenic as control BacPh OBs. The expression of En3 in the soluble fraction of insect cells may find applications in the development of virus-based insecticides with increased efficacy.

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