Chimeric Ad5/F35 Adenovirus Gene Expression Vector

Recombinant adenoviral vectors are used for achieving high levels of transgene expression in a wide variety of mammalian cell types, where the vector remains as episomal DNA without integrating into the host genome. High transduction efficiency and high levels of short-term gene expression make adenoviral vectors the preferred gene delivery tools for gene therapy and vaccine research.

Adenoviral vectors are derived from adenovirus, which causes the common cold. Wildtype adenovirus has a double-stranded linear DNA genome.

While human adenovirus serotype 5 (Ad5) vectors are the most widely used, a major disadvantage 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 chimeric Ad5/F35 adenoviral vectors to readily transduce CAR-negative cells in addition to CAR-positive cells, by exploiting the ability of Ad35 fiber protein to attach to target cells via a 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.

An adenoviral vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus. When the virus is added to target cells, the DNA cargo is delivered into cells where it enters the nucleus and remains as episomal DNA without integration into the host genome. Any gene(s) that were placed in-between the two ITRs during vector cloning are introduced into target cells along with the rest of viral genome.

By design, adenoviral vectors lack the E1A, E1B and E3 genes (delta E1 + delta E3). The first two are required for the production of live virus (these two genes are engineered into the genome of packaging cells). As a result, virus produced from the vectors have the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

For further information about this vector system, please refer to the papers below.

Our Ad5/F35 vector contains a chimeric fiber protein consisting of the knob and shaft derived from Ad35 and the fiber tail derived from Ad5 which enables it to have an expanded tropism compared to conventional Ad5 vectors, specifically for cells lacking or having insufficient expression of CAR. It is optimized for high-titer packaging of live virus, efficient transduction of host cells, and high-level transgene expression.

Expanded tropism: The Ad5/F35 vectors have an expanded tropism compared to Ad5 vectors, specifically for cells lacking or having insufficient expression of CAR. The presence of a chimeric fiber protein consisting of the knob and shaft derived from Ad35 on Ad5/F35 vectors enables them to infect CAR-negative cells by utilizing the CD46 receptor, unlike Ad5 vectors. 

Large cargo space: The Ad5/F35 vector has a larger cargo carrying capacity compared to the Ad5 vector due to the presence of the smaller fiber shaft coding sequence derived from Ad35 in these vectors. As a result, our Ad5/F35 vector has about ~8.2 kb of cargo space to accommodate the user's DNA of interest (such as promoter, ORF, and fluorescence marker) which is sufficient for most applications.

Low risk of host genome disruption: Upon transduction into host cells, adenoviral vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.

Very high viral titer: After our adenoviral vector is transfected into packaging cells to produce live virus, the virus can be further amplified to very high titer by re-infecting packaging cells. This is unlike lentivirus, MMLV retrovirus, or AAV, which cannot be amplified by re-infection. When adenovirus is obtained through our virus packaging service, titer can reach >10¹² VP/ml.

Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro.

Safety: The safety of our vector is ensured by the fact that it lacks genes essential for virus production (these genes are engineered into the genome of packaging cells). Virus made from our vector is therefore replication incompetent except when it is used to transduce packaging cells.

Non-integration of vector DNA: The adenoviral genome does not integrate into the genome of transduced cells. Rather, it exists as episomal DNA, which can be lost over time, especially in dividing cells.

Strong immunogenicity: Live virus from adenoviral vectors can elicit strong immune response in animals, thus limiting certain in vivo applications.

Technical complexity: The use of adenoviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming.

5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.

Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.

Promoter: The promoter that drives your gene of interest is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

BGH pA: Bovine growth hormone polyadenylation. It facilitates transcriptional termination of the upstream ORF.

hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression the downstream marker gene.

Marker: A visually detectable gene (such as EGFP). This allows cells transduced with the vector to be selected and/or visualized.

TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

ΔAd5/F35: Portion of adenovirus serotype 5 genome with adenovirus serotype 35 fiber shaft and knob between the two ITRs minus the E1A, E1B and E3 regions; exhibits expanded tropism, especially for cells either completely lacking or having insufficient coxsackievirus and adenovirus receptor (CAR) expression.

3' ITR: 3' inverted terminal repeat.

pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.