Adenovirus Packaging

Adenovirus production and quality control (QC)

For our adenovirus manufacturing, the adenoviral vector carrying the gene of interest (GOI) is first linearized by restriction digestion with PacI. The linearized vector DNA is transfected into packaging cells expressing the adenovirus gene E1 to produce recombinant adenovirus, and adenoviral particles are released into tissue culture medium where they are collected. For ultra-purified adenovirus (in vivo grade), viral particles are further purified and concentrated by cesium chloride (CsCl) gradient ultracentrifugation. We measure the titer using an immunocytochemistry-based approach to detect the adenovirus-specific hexon protein. For ultra-purified adenovirus, we measure the optical density (using OD260) of the viral particles to estimate the titer.

Figure 1. Typical workflow of Ad5 adenovirus packaging.

For each adenovirus produced by VectorBuilder, quality control includes titer measurement, sterility testing for bacteria and fungi, and mycoplasma detection. If the adenoviral vector encodes a fluorescent protein, we perform a transduction test to detect fluorescence. Additionally, for ultra-purified adenovirus, we routinely check endotoxin levels, the results of which can be included in your COA for an additional cost. Additional QC services can also be provided upon request.

For help determining which adenovirus type best suits your experimental needs, please refer to our FAQs.

Chimeric Ad5/F35 adenovirus production and quality control (QC)

For our chimeric Ad5/F35 adenovirus manufacturing (Figure 2), the Ad5/F35 adenoviral vector carrying the gene of interest (GOI) is first linearized by restriction digestion with PacI. The linearized vector DNA is transfected into packaging cells expressing the adenovirus gene E1 to produce recombinant adenovirus, and adenoviral particles are released into tissue culture medium where they are collected. For ultra-purified Ad5/F35 adenovirus (in vivo grade), viral particles are further purified and concentrated by cesium chloride (CsCl) gradient ultracentrifugation. We measure the titer using an immunocytochemistry-based approach to detect the adenovirus-specific hexon protein. For ultra-purified Ad5/F35 adenovirus, we measure the optical density (using OD260) of the viral particles to estimate the titer.

Figure 2. Typical workflow of chimeric Ad5/F35 adenovirus packaging.

For each adenovirus produced by VectorBuilder, quality control includes titer measurement, sterility testing for bacteria and fungi, and mycoplasma detection. If the Ad5/F35 adenoviral vector encodes a fluorescent protein, we perform a transduction test to detect fluorescence. Additionally, for ultra-purified adenovirus, we routinely check endotoxin levels, the results of which can be included in your COA for an additional cost. Additional QC services can also be provided upon request.

Ad5 adenovirus vs. chimeric Ad5/F35 adenovirus

While human adenovirus serotype 5 (Ad5) vectors are most widely used for adenovirus-based applications, a major limitation associated with such vectors is their dependence on the coxsackie and adenovirus receptor (CAR) for infecting target cells. Host cells that completely lack or have insufficient expression of CAR cannot be efficiently transduced with Ad5 vectors. Ad5/F35 vectors help overcome this limitation by expressing a chimeric fiber protein comprised of the knob and shaft derived from adenovirus serotype 35 (Ad35) and the fiber tail derived from Ad5. This enables Ad5/F35 adenoviral vectors to readily transduce CAR-negative cells or cells with low levels of CAR-expression in addition to CAR-positive cells with high levels of CAR expression, by exploiting the ability of the Ad35 fiber protein to attach to target cells via the non-CAR receptor, CD46. The chimeric design of Ad5/F35 adenovirus has been highly instrumental in expanding the tropism of adenoviral vectors to cell lines such as hematopoietic cells, primitive stem cells, vascular smooth muscle cells and CAR-negative tumor cells which otherwise exhibit poor transduction efficiency with conventional Ad5 vectors.

For help determining which adenovirus type best suits your experimental needs, please refer to our FAQs.

Gutless Ad5 adenovirus production and quality control (QC)

For our gutless adenovirus manufacturing (Figure 3), an expression cassette containing the user-selected gene of interest (GOI) driven by a promoter is first cloned into our gutless adenovirus vector in between the two inverted terminal repeats (ITRs). Additionally, our vector incorporates stuffer sequences of appropriate lengths for maintaining a final size of above 28 kb between the two ITRs, which is necessary to facilitate the efficient packaging of virions. The region of the vector flanked by the two ITRs is then released from the plasmid by restriction digestion. The released fragment is transfected into packaging cells which are subsequently infected with the helper virus to generate recombinant adenoviral particles. Our gutless adenoviral vector system utilizes a helper virus with the packaging signal flanked by two LoxP sites along with packaging cells which stably express Cre recombinase facilitating Cre-mediated excision of the helper virus packaging signal. This prevents the helper virus genome from being packaged into viral particles along with the gutless adenoviral genome. Adenoviral particles are released into tissue culture medium where they are collected.

For ultra-purified gutless adenovirus (in vivo grade), viral particles are further purified and concentrated by cesium chloride (CsCl) gradient ultracentrifugation. We measure the optical density (using OD260) of the viral particles to estimate the titer.

Figure 3. Typical workflow of gutless Ad5 adenovirus packaging.

For each adenovirus produced by VectorBuilder, quality control includes titer measurement, sterility testing for bacteria and fungi, and mycoplasma detection. If the gutless adenoviral vector encodes a fluorescent protein, we perform a transduction test to detect fluorescence. Additionally, for ultra-purified adenovirus, we routinely check endotoxin levels, the results of which can be included in your COA for an additional cost. Additional QC services can also be provided upon request.

Advantages of gutless adenovirus over Ad5 and Ad/F35 adenovirus

Gutless adenoviral vectors (a.k.a. helper-dependent adenoviral vectors) are the latest generation of adenoviral vectors which offer several advantages over conventional Ad5 or chimeric Ad5/F35 adenoviral vectors. The absence of nearly all viral sequences on these vectors except cis-acting elements essential for viral replication and packaging enables gutless adenoviral vectors to exhibit minimal immunogenicity and prolonged transgene expression when used in vivo. Therefore, they have a significantly improved safety profile compared to Ad5 or Ad5/F35 which makes them highly attractive candidates for gene therapy applications. Additionally, the lack of viral coding sequences allows them to have an increased cargo carrying capacity of up to 33 kb, making them highly suitable for the expression of long or multiple transgenes. 

For help determining which adenovirus type best suits your experimental needs, please refer to our FAQs.

Gutless Ad5/F35 adenovirus production and quality control (QC)

For our gutless Ad5/F35 adenovirus manufacturing (Figure 4), an expression cassette containing the user-selected gene of interest (GOI) driven by a promoter is first cloned into our gutless adenovirus vector in between the two inverted terminal repeats (ITRs). Additionally, our vector incorporates stuffer sequences of appropriate lengths for maintaining a final size of above 28 kb between the two ITRs, which is necessary to facilitate the efficient packaging of virions. The region of the vector flanked by the two ITRs is then released from the plasmid by restriction digestion. The released fragment is transfected into packaging cells which are subsequently infected with the helper virus to generate recombinant adenoviral particles. Our gutless adenoviral vector system utilizes a helper virus with the packaging signal flanked by two LoxP sites along with packaging cells which stably express Cre recombinase, facilitating Cre-mediated excision of the helper virus packaging signal. This prevents the helper virus genome from being packaged into viral particles along with the gutless adenoviral genome. Adenoviral particles are released into the tissue culture medium where they are collected.

For ultra-purified gutless adenovirus (in vivo grade), viral particles are further purified and concentrated by cesium chloride (CsCl) gradient ultracentrifugation. We measure the optical density (using OD260) of the viral particles to estimate the titer.

Figure 4. Typical workflow of gutless Ad5/F35 adenovirus packaging.

For each adenovirus produced by VectorBuilder, quality control includes titer measurement, sterility testing for bacteria and fungi, and mycoplasma detection. If the gutless adenoviral vector encodes a fluorescent protein, we perform a transduction test to detect fluorescence. Additionally, for ultra-purified adenovirus, we routinely check endotoxin levels, the results of which can be included in your COA for an additional cost. Additional QC services can also be provided upon request.

For help determining which adenovirus type best suits your experimental needs, please refer to our FAQs.

Advantages of gutless Ad5/F35 adenovirus over gutless Ad5 and conventional adenovirus

Our Ad5/F35 gutless adenoviral vectors combine the advantages of both gutless adenovirus and Ad/F35 adenovirus, exhibiting reduced immunogenicity, prolonged transgene expression, greater carrying capacity, and a broader tropism.

Gutless adenoviral vectors (a.k.a. helper-dependent adenoviral vectors) are the latest generation of adenoviral vectors which offer several advantages over conventional adenoviral vectors. The absence of nearly all viral sequences in these vectors, except cis-acting elements essential for viral replication and packaging, enables gutless adenoviral vectors to exhibit minimal immunogenicity and prolonged transgene expression when used in vivo. Therefore, they have a significantly improved safety profile compared to conventional Ad5 or Ad5/F35, which makes them attractive candidates for gene therapy applications. Additionally, the lack of viral coding sequences allows them to have an increased cargo carrying capacity of up to 33 kb, making them highly suitable for the expression of long or multiple transgenes.

Human adenovirus serotype 5 (Ad5) vectors are most widely used in adenovirus-based applications; however, their effectiveness is often limited by their reliance on the coxsackie and adenovirus receptor (CAR) to infect target cells. Ad5/F35 vectors address this limitation by incorporating a chimeric fiber protein, which combines the knob and shaft from adenovirus serotype 35 (Ad35) with the fiber tail from Ad5. This design allows Ad5/F35 vectors to efficiently transduce not only CAR-positive cells but also CAR-negative cells, or those with low CAR expression, by exploiting the ability of the Ad35 fiber protein to attach to target cells via the non-CAR receptor, CD46. The chimeric design of Ad5/F35 adenovirus has been instrumental in expanding the tropism of adenoviral vectors to cell lines such as hematopoietic cells (HSCs), primitive stem cells, vascular smooth muscle cells and CAR-negative tumor cells which otherwise exhibit poor transduction efficiency with conventional Ad5 vectors.