Plasmid transfection of mammalian cells is the dominant platform used to produce adeno-associated virus (AAV) vectors for clinical and research applications.
Low yields from this platform currently make it difficult to supply these activities with adequate material. In an effort to better understand the current limitations of transfection-based manufacturing, this study examines what proportion of cells in a model transfection produce appreciable amounts of assembled AAV capsid.
Using conformation-specific antibody staining and flow cytometry we report the surprising result that despite obtaining high transfection efficiencies and nominal vector yields in our model system, only 5-10% of cells appear to produce measurable levels of assembled AAV capsids.
This finding implies that considerable increases in vector titer could be realized through increasing the proportion of productive cells.
Furthermore, we suggest that the flow cytometry assay used here to quantify productive cells may be a useful metric for future optimization of transfection-based AAV vector manufacturing platforms.
This article is protected by copyright. All rights reserved.
Virus-Induced Gene Silencing in Chrysanthemum seticuspe Using the Tomato Aspermy Virus Vector.
Chrysanthemum is one of the most economically important flowers globally due to its high ornamental value. In recent years, a large percentage of the Chrysanthemum seticuspe genome has been determined, making this species useful as a model chrysanthemum plant.
To fully utilize the genome’s information, efficient and rapid gene functional analysis methods are needed. In this study, we optimized the tomato aspermy virus (TAV) vector for virus-induced gene silencing (VIGS) in C. seticuspe.
Conventional plant virus inoculation methods, such as the mechanical inoculation of viral RNA transcripts and agroinoculation into leaves, did not achieve successful TAV infections in C. seticuspe, but vacuum infiltration into sprouts was successful without symptoms.
The TAV vector harboring 100 nucleotides of the phytoene desaturase (PDS) gene caused photobleaching phenotypes and a reduction in CsPDS expression in C. seticuspe. To our knowledge, this is the first report of VIGS in chrysanthemums.
Therapeutic Application of Extracellular Vesicles-Capsulated Adeno-Associated Virus Vector via nSMase2/Smpd3, Satellite, and Immune Cells in Duchenne Muscular Dystrophy.
- Duchenne muscular dystrophy (DMD) is caused by loss-of-function mutations in the dystrophin gene on chromosome Xp21. Disruption of the dystrophin-glycoprotein complex (DGC) on the cell membrane causes cytosolic Ca2+ influx, resulting in protease activation, mitochondrial dysfunction, and progressive myofiber degeneration, leading to muscle wasting and fragility.
- In addition to the function of dystrophin in the structural integrity of myofibers, a novel function of asymmetric cell division in muscular stem cells (satellite cells) has been reported.
- Therefore, it has been suggested that myofiber instability may not be the only cause of dystrophic degeneration, but rather that the phenotype might be caused by multiple factors, including stem cell and myofiber functions.
- Furthermore, it has been focused functional regulation of satellite cells by intracellular communication of extracellular vesicles (EVs) in DMD pathology.
- Recently, a novel molecular mechanism of DMD pathogenesis-circulating RNA molecules-has been revealed through the study of target pathways modulated by the Neutral sphingomyelinase2/Neutral sphingomyelinase3 (nSMase2/Smpd3) protein.
- In addition, adeno-associated virus (AAV) has been clinically applied for DMD therapy owing to the safety and long-term expression of transduction genes.
- Furthermore, the EV-capsulated AAV vector (EV-AAV) has been shown to be a useful tool for the intervention of DMD, because of the high efficacy of the transgene and avoidance of neutralizing antibodies. Thus, we review application of AAV and EV-AAV vectors for DMD as novel therapeutic strategy.
Carotenoid fortification of zucchini fruits using a viral RNA vector.
Carotenoids are health-promoting metabolites in livestock and human diets. Some important crops have been genetically modified to increase their content.
Although the use of transgenic plants to alleviate nutritional deficiencies is obvious, their social acceptance has been controversial.
Here, we demonstrate an alternative biotechnological strategy for carotenoid fortification of edible fruits in which no transgenic DNA is involved.
A viral RNA vector derived from Zucchini yellow mosaic virus (ZYMV) was modified to express a bacterial phytoene synthase (crtB), and inoculated to zucchini (Cucurbita pepo L.) leaves nurturing pollinated flowers.
After the viral vector moved to the developing fruit and expressed crtB, the rind and flesh of the fruits developed yellow-orange rather than green color.
Metabolite analyses showed a substantial enrichment in health-promoting carotenoids, such as α- and β-carotene (provitamin A), lutein and phytoene, in both rind and flesh.
Although this strategy is perhaps not free from controversy due to the use of genetically modified viral RNA, our work does demonstrate the possibility of metabolically fortifying edible fruits using an approach in which no transgenes are involved. This article is protected by copyright. All rights reserved.
COVID-19 vaccine development based on recombinant viral and bacterial vector systems: combinatorial effect of adaptive and trained immunity.
Severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) infection, which causes coronavirus disease 2019 (COVID-19), has led to many cases and deaths worldwide. Therefore, a number of vaccine candidates have been developed to control the COVID-19 pandemic.
Of these, to date, 21 vaccines have received emergency approval for human use in at least one country. However, the recent global emergence of SARS-CoV-2 variants has compromised the efficacy of the currently available vaccines.
To protect against these variants, the use of vaccines that modulate T cell-mediated immune responses or innate immune cell memory function, termed trained immunity, is needed.
The major advantage of a vaccine that uses bacteria or viral systems for the delivery of COVID-19 antigens is the ability to induce both T cell-mediated and humoral immune responses.
In addition, such vaccine systems can also exert off-target effects via the vector itself, mediated partly through trained immunity; compared to other vaccine platforms, suggesting that this approach can provide better protection against even vaccine escape variants.
Green Kit. Baculovirus GFP vector. |
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| K20 | AB Vector LLC | 1 Kit | 695 EUR |
ProGreen. Baculovirus GFP marker vector. |
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| A1 | AB Vector LLC | 25 ul | 420 EUR |
pVL1393. General baculovirus plasmid vector. |
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| B1 | AB Vector LLC | 50 ul | 340 EUR |
ProEasy. Vector for easy construction of recombinant baculoviruses. |
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| A10S | AB Vector LLC | 25 ul | 695 EUR |
pAcAB3. Baculovirus plasmid vector for expression of up to 3 proteins. |
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| B2 | AB Vector LLC | 50 ul | 420 EUR |
pAB-bee. Baculovirus plasmid vector for secreted and transmembrane proteins. |
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| B3 | AB Vector LLC | 50 ul | 495 EUR |
ProFold-PDI. Baculovirus chaperone vector for expression of cysteine-rich proteins. |
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| A7 | AB Vector LLC | 25 ul | 830 EUR |
ProFold-C1. Baculovirus chaperone vector for expression of cytoplasmic and nuclear proteins. |
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| A2 | AB Vector LLC | 25 ul | 830 EUR |
ProFold-C2. Baculovirus chaperone vector for expression of cytoplasmic and nuclear proteins. |
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| A3 | AB Vector LLC | 25 ul | 830 EUR |
ProFold-ER1. Baculovirus chaperone vector for expression of secreted and membrane proteins. |
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| A4 | AB Vector LLC | 25 ul | 830 EUR |
C1 Kit. Baculovirus chaperone vectors for cytoplasmic and nuclear proteins. |
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| K21 | AB Vector LLC | 1 Kit | 995 EUR |
C2 Kit. Baculovirus chaperone vectors for cytoplasmic and nuclear proteins. |
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| K22 | AB Vector LLC | 1 Kit | 995 EUR |
ER1 Kit. Baculovirus chaperone vectors for expression of secreted and membrane proteins. |
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| K23 | AB Vector LLC | 1 Kit | 995 EUR |
ER1-bee Kit. Baculovirus chaperone vectors for expression of secreted and membrane proteins. |
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| K24 | AB Vector LLC | 1 Kit | 995 EUR |
pAB-bee-FH. FLAG and His tagging of secreted and transmembrane proteins in baculovirus system. |
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| B3FH | AB Vector LLC | 50 ul | 495 EUR |
pAB-bee-8xHis. His tagging of secreted and transmembrane proteins in baculovirus system. |
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| B3H | AB Vector LLC | 50 ul | 495 EUR |
pAB-6xHis. His tagging of cytoplasmic and nuclear proteins in baculovirus system. |
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| B4 | AB Vector LLC | 50 ul | 295 EUR |
This review presents the current status of the development of COVID-19 vaccines based on recombinant viral and bacterial delivery systems. We also discuss the current status of the use of licensed live vaccines for other infections, including BCG, oral polio and MMR vaccines, to prevent COVID-19 infections.