Adenovirus Vector Information

Adenovirus Vector Information : Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. Homologous recombination in E. coli between shuttle plasmids and full-length adenovirus backbones (E1-deleted) is used for the generation of recombinant adenoviral vectors genomes. The adenovirus genomes are then analyzed to confirm their identity and integrity, and further linearized and transfected to generate a recombinant adenoviral vector in permissive human cells. These vectors are then purified by two sequential CsCl gradient centrifugations and subjected to a chromatography step in order to eliminate the CsCl and exchange buffers. Finally, the viral stock is characterized through the quantification of its viral particle content and its infectivity.

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– Tropism: wide; preferent for CAR-expressing cells.


– Common Applications: short term in vivo studies.


– Procedure:
– Cloning in shuttle plasmid / homologous recombination.
– HEK-293 cells transfection.
– Amplification to 4x10E8 HEK-293 cells (standard production).
– Vector Purification: double cesium chloride gradient/ gel filtration chromatography.


– Characterization:
– Physical Particle (PP) quantification.
– Infectious particle (IU) quantification.


– Quality Control:
– Sterility tests: Tryptic Soy Broth and Fluid Thioglycollated Medium prepared according to the recommendations of the current European and United States Pharmacopeia.
– Mycoplasm test.
– RCA detection (under request).


– Average total yields (hAd): 4x10E12 PP, 3x10E11 IU


– Average titers (hAd): 1,5x10E12 PP/mL, 1x10E11 IU/mL


– Production time of AAV Vector (without cloning): 1,5 months.

The tropism of each Adenovirus serotype is indicated in the chart below.

Tissue
Optimal Serotype
CAR receptors
Ad5
Macrophages, monocytes, intestinal cells
Ad5/40
Neuronal origin cells
Ad5/52
Neurons, respiratory cells
CAV
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