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International Journal of Molecular... Feb 2024Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However,...
A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency.
Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.
Topics: Animals; Capsid Proteins; Dependovirus; Chromatography, Liquid; Liquid Chromatography-Mass Spectrometry; Workflow; Genetic Vectors; Tandem Mass Spectrometry; Baculoviridae; Mammals
PubMed: 38474031
DOI: 10.3390/ijms25052785 -
Nucleic Acids Research Apr 2024CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components...
CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes.
Topics: Animals; Humans; Baculoviridae; CRISPR-Associated Protein 9; CRISPR-Cas Systems; DNA; Gene Editing; Viral Proteins; Cell Line
PubMed: 38412306
DOI: 10.1093/nar/gkae142 -
Viruses Feb 2024, a virus family characterized by a single large double stranded DNA, encompasses the majority of viral bioinsecticides, representing a highly promising and...
, a virus family characterized by a single large double stranded DNA, encompasses the majority of viral bioinsecticides, representing a highly promising and environmentally friendly pesticide approach to insect control. This study focuses on the characterization of a baculovirus isolated from larvae of (Erebidae, Lymantriidae) collected in Mongolian pinaceae forests. This new isolate was called nucleopolyhedrovirus (CaabNPV). CaabNPV exhibits an irregular polyhedron shape, and significant variation in the diameter of its occlusion bodies (OBs) was observed. Nucleotide distance calculations confirmed CaabNPV as a novel baculovirus. The CaabNPV genome spans 177,161 bp with a G+C content of 45.12% and harbors 150 potential open reading frames (ORFs), including 38 core genes. A comprehensive genomic analysis categorizes CaabNPV within Group II alphabaculovirus, revealing a close phylogenetic relationship with (OrleNPV). Additionally, repeat sequence analysis identified three highly repetitive sequences consisting of 112 bp repeat units, known as homologous regions (). This research contributes valuable insights into CaabNPV's phylogenetic placement, genomic structure, and its potential applications in insect biocontrol.
Topics: Animals; Nucleopolyhedroviruses; Baculoviridae; Phylogeny; Genome, Viral; Moths; Sequence Analysis, DNA; Open Reading Frames
PubMed: 38400028
DOI: 10.3390/v16020252 -
Journal of Innate Immunity 2024The brain is considered as an immune-privileged organ, yet innate immune reactions can occur in the central nervous system of vertebrates and invertebrates. Silkworm...
INTRODUCTION
The brain is considered as an immune-privileged organ, yet innate immune reactions can occur in the central nervous system of vertebrates and invertebrates. Silkworm (Bombyx mori) is an economically important insect and a lepidopteran model species. The diversity of cell types in the silkworm brain, and how these cell subsets produce an immune response to virus infection, remains largely unknown.
METHODS
Single-nucleus RNA sequencing (snRNA-seq), bioinformatics analysis, RNAi, and other methods were mainly used to analyze the cell types and gene functions of the silkworm brain.
RESULTS
We used snRNA-seq to identify 19 distinct clusters representing Kenyon cell, glial cell, olfactory projection neuron, optic lobes neuron, hemocyte-like cell, and muscle cell types in the B. mori nucleopolyhedrovirus (BmNPV)-infected and BmNPV-uninfected silkworm larvae brain at the late stage of infection. Further, we found that the cell subset that exerts an antiviral function in the silkworm larvae brain corresponds to hemocytes. Specifically, antimicrobial peptides were significantly induced by BmNPV infection in the hemocytes, especially lysozyme, exerting antiviral effects.
CONCLUSION
Our single-cell dataset reveals the diversity of silkworm larvae brain cells, and the transcriptome analysis provides insights into the immune response following virus infection at the single-cell level.
Topics: Animals; Bombyx; Brain; Larva; Hemocytes; Immunity, Innate; Muramidase; Nucleopolyhedroviruses; Single-Cell Analysis; Insect Proteins
PubMed: 38387449
DOI: 10.1159/000537815 -
Open Biology Feb 2024Neuroparasitism concerns the hostile take-over of a host's nervous system by a foreign invader, in order to alter the behaviour of the host in favour of the parasite....
Neuroparasitism concerns the hostile take-over of a host's nervous system by a foreign invader, in order to alter the behaviour of the host in favour of the parasite. One of the most remarkable cases of parasite-induced host behavioural manipulation comprises the changes baculoviruses induce in their caterpillar hosts. Baculoviruses may manipulate caterpillar behaviour in two ways: hyperactivity (increased movement in the horizontal plane) and/or tree-top disease (movement to elevated levels in the vertical plane). Those behavioural changes are followed by liquefaction and death of the caterpillar. In Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected caterpillars, an enzymatic active form of the virally encoded protein tyrosine phosphatase (PTP) is needed for the expression of hyperactivity from 3 days post infection (dpi). Using eGFP-expressing recombinant AcMNPV strains, we show that infection of the caterpillar's central nervous system (CNS) can be observed primarily from 3 dpi onwards. In addition, we demonstrate that the structural and enzymatic function of PTP does not play a role in infection of the CNS. Instead we show that the virus entered the CNS via the trachea, progressing caudally to frontally through the CNS and that the infection progressed from the outermost cell layers towards the inner cell layers of the CNS, in a PTP independent manner. These findings help to further understand parasitic manipulation and the mechanisms by which neuroparasites infect the host nervous system to manipulate host behaviour.
Topics: Animals; Baculoviridae; Spodoptera; Central Nervous System; Protein Tyrosine Phosphatases; Nucleopolyhedroviruses
PubMed: 38378139
DOI: 10.1098/rsob.230278 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Feb 2024Adeno-associated virus (AAV) is one of the most frequently used viral vectors in the field of gene therapy. However, the industrial production of AAV is facing key...
Adeno-associated virus (AAV) is one of the most frequently used viral vectors in the field of gene therapy. However, the industrial production of AAV is facing key bottlenecks such as low yield and high-cost. The aim of this study was to establish a technology system for production of AAV in the double virus infected insects by using multiple-gene deleted baculovirus. First, a multiple gene deleted baculovirus for AAV production was constructed, and the baculovirus titer and its effect on infected cells was examined. Subsequently, the insect cells were co-infected with the double baculovirus and the infection conditions were optimized. At the final stage, we performed AAV production based on optimized conditions, and evaluated relevant parameters including production titer and quality. The results showed that the titer of AAV produced in the multiple gene deleted baculovirus was not different from that of the wild type, but the rate of cell death was significantly slower upon infection. Using the double virus route for optimized production of AAV, the genome titers were 1.63×10 VG/mL for Bac4.0-1 and 1.02×10 VG/mL for Bac5.0-2, which were elevated 240% and 110%, respectively, compared with that of the wild-type. Electron microscopy observations revealed that all three groups exhibited normal AAV viral morphology and they showed similar transduction activity. Taken together, we developed an AAV production system based on the infection of insect cells using multiple-gene deleted baculovirus, which significantly improved the virus yield and showed application potential.
Topics: Animals; Dependovirus; Baculoviridae; Cell Line; Genetic Vectors; Insecta
PubMed: 38369834
DOI: 10.13345/j.cjb.230325 -
New Biotechnology May 2024The aim of this study was the development of a scalable production process for high titer (10 pfu/mL and above) recombinant baculovirus stocks with low cell line-derived...
The aim of this study was the development of a scalable production process for high titer (10 pfu/mL and above) recombinant baculovirus stocks with low cell line-derived impurities for the production of virus-like particles (VLP). To achieve this, we developed a high cell density (HCD) culture for low footprint cell proliferation, compared different infection strategies at multiplicity of infection (MOI) 0.05 and 0.005, different infection strategies and validated generally applicable harvest criteria of cell viability ≤ 80%. We also investigated online measurable parameters to observe the baculovirus production. The infection strategy employing a very low virus inoculum of MOI 0.005 and a 1:2 dilution with fresh medium one day after infection proved to be the most resource efficient. There, we achieved higher cell-specific titers and lower host cell protein concentrations at harvest than other tested infection strategies with the same MOI, while saving half of the virus stock for infecting the culture compared to other tested infection strategies. HCD culture by daily medium exchange was confirmed as suitable for seed train propagation, infection, and baculovirus production, equally efficient as the conventionally propagated seed train. Online measurable parameters for cell concentration and average cell diameter were found to be effective in monitoring the production process. The study concluded that a more efficient VLP production process in large scale can be achieved using this virus stock production strategy, which could also be extended to produce other proteins or extracellular vesicles with the baculovirus expression system.
Topics: Baculoviridae; Cell Line; Cell Proliferation; Cell Count
PubMed: 38302001
DOI: 10.1016/j.nbt.2024.01.002 -
Viruses Jan 2024Metagenomic analysis of and mosquitoes from diverse geographical regions of India revealed the presence of several insect viruses of human interest. Most abundant...
Metagenomic analysis of and mosquitoes from diverse geographical regions of India revealed the presence of several insect viruses of human interest. Most abundant reads found in mosquitoes were of Phasi Charoen-like virus (PCLV), granulovirus (CfGV), Cell fusing agent virus (CFAV), and Wenzhou sobemo-like virus 4 (WSLV4), whereas WSLV4 and CfGV constituted the highest percentage of reads in viromes. Other reads that were of low percentage included Hubei mosquito virus 2 (HMV2), Porcine astrovirus 4 (PAstV4), and Wild Boar astrovirus (WBAstV). PCLV and CFAV, which were found to be abundant in . viromes were absent in viromes. Among the viromes analyzed, sampled from Pune showed the highest percentage (79.82%) of viral reads, while . mosquitoes sampled from Dibrugarh showed the lowest percentage (3.47%). Shamonda orthobunyavirus (SHAV), African swine fever virus (ASFV), Aroa virus (AROAV), and Ilheus virus (ILHV), having the potential to infect vertebrates, including humans, were also detected in both mosquito species, albeit with low read numbers. Reads of gemykibivirus, avian retrovirus, bacteriophages, herpesviruses, and viruses infecting protozoans, algae, etc., were also detected in the mosquitoes. A high percentage of reads in the mosquito samples belonged to unclassified viruses and warrant further investigation. The data generated in the present work may not only lead to studies to explain the influence of these viruses on the replication and transmission of viruses of clinical importance but also to find applications as biocontrol agents against pathogenic viruses.
Topics: Animals; Swine; Humans; Aedes; African Swine Fever Virus; Virome; India; Bacteriophages; Arenaviridae; Granulovirus
PubMed: 38257809
DOI: 10.3390/v16010109 -
Viruses Dec 2023The baculovirus expression vector (BEV) system is an efficient, cost-effective, and scalable method to produce recombinant adeno-associated virus (rAAV) gene therapy...
The baculovirus expression vector (BEV) system is an efficient, cost-effective, and scalable method to produce recombinant adeno-associated virus (rAAV) gene therapy vectors. Most BEV designs emulate the wild-type AAV transcriptome and translate the AAV capsid proteins, VP1, VP2, and VP3, from a single mRNA transcript with three overlapping open reading frames (ORFs). Non-canonical translation initiation codons for VP1 and VP2 reduce their abundances relative to VP3. Changing capsid ratios to improve rAAV vector efficacy requires a theoretical modification of the translational context. We have developed a Lac repressor-inducible system to empirically regulate the expression of VP1 and VP2 proteins relative to VP3 in the context of the BEV. We demonstrate the use of this system to tune the abundance, titer, and potency of a neurospecific rAAV9 serotype derivative. VP1:VP2:VP3 ratios of 1:1:8 gave optimal potency for this rAAV. It was discovered that the ratios of capsid proteins expressed were different than the ratios that ultimately were in purified capsids. Overexpressed VP1 did not become incorporated into capsids, while overexpressed VP2 did. Overabundance of VP2 correlated with reduced rAAV titers. This work demonstrates a novel technology for controlling the production of rAAV in the BEV system and shows a new perspective on the biology of rAAV capsid assembly.
Topics: Capsid; Capsid Proteins; Dependovirus; Lac Repressors; Baculoviridae
PubMed: 38257750
DOI: 10.3390/v16010051 -
Genes Dec 2023The resistance of silkworms to nuclear polyhedrosis virus (BmNPV) is controlled by a major dominant gene and multiple modifying genes. Given the presence of modified...
The resistance of silkworms to nuclear polyhedrosis virus (BmNPV) is controlled by a major dominant gene and multiple modifying genes. Given the presence of modified genes, it is difficult to determine the main gene by positional cloning. In this study, the main anti-BmNPV gene of BmNPV-resistant silkworm variety N was introduced into the susceptible variety Su to breed the near-isogenic line SuN with BmNPV resistance. The infection process of BmNPV in the hemolymph of Su and SuN was analyzed using the cell analysis system TissueFAXS PLUS. According to the law of infection and proliferation, hemolymph was extracted every 6 h for two-dimensional electrophoresis (2-DE) analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Seven DEPs were found in comparisons between Su and SuN by 2-DE analysis. Among them, acid phosphatase, storage protein, and phenoloxidase can prevent pathogen invasion, which may play a role against BmNPV. Polyamine oxidase plays an important role in energy metabolism, which may be indirectly involved in the process of resisting BmNPV. Most of the transcriptional expression profiles of the seven DEPs were consistent with the 2-DE results. This study can provide a reference for the identification of anti-BmNPV genes and the breeding of BmNPV-resistant silkworm varieties.
Topics: Animals; Bombyx; Nucleopolyhedroviruses; Proteomics; Genes, Dominant
PubMed: 38254949
DOI: 10.3390/genes15010059