Custom Adenoviral Vector Production Service

Overview of Adenoviral Vector

Adenovirus (AD) is a non-enveloped linear double-stranded DNA virus that does not integrate cellular DNA (maintains dsDNA episomes within cells), can carry large fragments of foreign DNA, infect dividing and non-dividing cells, and induce humoral and cellular immune responses (which is helpful for the development of vaccines). These characteristics make adenoviruses valuable as vaccination, oncolytic virus therapy, and transgenic vectors for gene therapy.

Adenoviral vectors can transduce a variety of cell types and have high transgene effectiveness (almost 100% transduction efficiency) when used in vitro. They are effective techniques for delivering genes to lots of cells that are challenging to transfect. However, due to the significant immunogenicity of adenovirus and the severe infection, when employed in vivo, it frequently results in inflammatory reactions and immunological responses in the local tissues of animals. Adenoviral vectors, in comparison to other viral vectors, have the following benefits:

• Express quickly. Adenovirus can be expressed in cells after 1-2 days after infection.
• Have a huge carrying capacity, and a high infection efficiency, and are frequently utilized to infect cells that are difficult to infect.
• No insertional mutagenicity, not incorporated into chromosomes.
• Easy to infect liver cells and hepatotropic. Adenoviruses are used in vivo to infect the liver.

Production

The first- and second-generation adenoviral vectors are disseminated using trans-complementing cell lines, such as the E1 complementing HEK 293 cell line. Contrarily, third-generation or helper-dependent (adeno)virus (HDV) vector propagation is reliant on the simultaneous infection of complementary cell lines with HDV and a helper virus, including the primary rescue of the HDV following transfection of HDV-DNA. Homologous recombination is frequently carried out to simplify the inclusion of a particular gene of interest into the viral vector. To make a recombinant adenoviral vector, a gene of interest is typically placed into the E1 region of an adenoviral vector backbone. For first-generation adenoviruses (E1/E3-deleted Ad5) and chimeric adenoviruses, we provide packaging services. The first-generation adenovirus packing technology that we usually employ consists of a Transfer plasmid with an adenovirus genome sequence with the E1/E3 gene deleted and an adenoviral E1-producing cell line expressing the E1 gene. The packaging process uses endolysis. The enzyme PacI linearizes the transfer plasmid that contains the GOI. The E1 gene's expression product stimulates the expression of the adenovirus gene on the transferred plasmid after linearized plasmid DNA has been transfected into packaging cells. This causes the production of recombinant adenovirus particles. Only basic adenovirus is currently produced. Again, crude adenovirus is transduced into packing cells for amplification.

Fig.1 Procedure of adenoviral vectors.

Purification

Adenovirus can be extracted from cells and added directly to the culture medium. Cesium chloride (CsCl) density centrifugation is necessary for additional blocking and concentration of ultra-blocked adenovirus. Please get in touch with us to find out more if you need any other purification techniques.

Fig.2 CsCl gradient-based virus purification.¹

Quality Control

Our quality control procedures for adenovirus include Titer determination using immunocytochemical staining (ICC), Bioburden tests, and Mycoplasma tests. Additional Endotoxin tests are used for ultra-purified adenovirus. Adenovirus will also undergo transduction tests if the transfer vector encodes the fluorescent protein.

The manufacturing and quality assurance processes for Creative Biolabs' adenovirus follow industry standards accepted by the global academic community. Adenovirus typically has a titer of 10¹⁰-10¹¹ pfu/ml, which can adequately satisfy the nee ds of diverse studies. Please contact us if you would like more information about our adenovirus production services.

Reference

1. Kim, Julius W et al. "Viral Vector Production: Adenovirus." Methods in molecular biology (Clifton, N.J.) vol. 1382 (2016): 115-30. doi:10.1007/978-1-4939-3271-9_9