Gutless Adenovirus Gene Expression Vector

Gutless adenoviral vectors (a.k.a. helper-dependent adenoviral vectors) are the latest generation of adenoviral vectors with a significantly improved safety profile compared to their earlier generations. 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, thereby making them highly attractive candidates for gene therapy. Additionally, the lack of viral coding sequences allows them to have an increased cargo carrying capacity of up to 33 kb, making them suitable for expression of long or multiple transgenes.

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

Since the gutless adenovirus gene expression vector is devoid of all viral coding sequences, viral proteins required for successful packaging of recombinant virus are supplied in trans by a helper virus. 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 ITRs. Additionally, our vector incorporates stuffer sequences of appropriate lengths for maintaining a final size of above 28 kb between the two inverted terminal repeats (ITRs), which is necessary to facilitate the efficient packaging of virions. The region of the vector flanked by the two ITRs is 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 to facilitate Cre-mediated excision of the helper virus packaging signal. This prevents the helper virus genome from being packaged into viral particles along with gutless adenoviral genome. To further minimize helper virus contamination, we recommend that the size of user defined sequences inserted between the two ITRs should be in the range of 8 to 12 kb. This would result in the final viral genome size to be in the range of 28 to 33 kb, which in turn would facilitate its separation from the helper virus genome during the purification process.

When the recombinant 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.

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

High safety: Our gutless adenoviral vectors are characterized by the absence of all viral sequences except cis-acting elements essential for viral replication and packaging, leading to a significant improvement in their safety profile compared to the previous generations of adenoviral vectors. As a result, such vectors offer the advantages of minimized host immune response and prolonged transgene expression in vivo.

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 >10¹⁰ infectious units per ml (IFU/ml).

Broad tropism: Cells from commonly used mammalian species such as human, mouse and rat can be transduced with our vector, but some cell types have proven difficult to transduce (see disadvantages below).

Large cargo space: The deletion of all viral coding sequences renders gutless adenoviral vectors with a large cargo carrying capacity, making them highly suitable for applications requiring expression of large or multiple transgenes. The recommended viral genome size for our gutless adenoviral vector is 28 to 33 kb, so that it can be efficiently packaged into adenovirus and easily separated from helper virus during purification process. This allows users to insert sequences of up to 8 to 12 kb in between the two ITRs, which is typically sufficient for most applications. 

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.

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.

Difficulty transducing certain cell types: While our adenoviral vectors can transduce many different cell types including non-dividing cells, it is inefficient against certain cell types such as endothelia, smooth muscle, differentiated airway epithelia, peripheral blood cells, neurons, and hematopoietic cells.

Helper virus contamination: Our gutless adenoviral vector system utilizes a helper virus containing a floxed packaging signal along with packaging cells which stably express Cre recombinase to restrict the helper virus genome from being packaged into viral particles along with the gutless adenoviral genome by Cre-mediated excision of the helper virus packaging signal. However, low levels of helper virus contamination might still be present in the recombinant viral preps due to inefficient excision of the packaging signal. The presence of such low levels of helper virus contamination might have associated toxicity effects at high doses of the recombinant virus.  

Technical complexity: The production and amplification of recombinant gutless adenoviral vectors is far more technically challenging compared to the earlier adenoviral vector generations because it involves the addition of the helper virus with each passage.

3' ITR: 3' 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.

Ad5_E4 fragment: Adenovirus serotype 5 E4 gene promoter region. Can improve vector stability and supports packaging.

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 signal. 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.

C346_Stuffer: Part of C346 cosmid (GenBank L31948) DNA sequence. Used for maintaining a final size above 28 kb between the two ITRs of vectors to facilitate efficient packaging into virions.

HPRT_Stuffer: Part of human hypoxanthine-guanine phosphoribosyltransferase (HPRT1) DNA sequence (containing introns 1, 2 and 3 and exon 2, 3). Used for maintaining a final size above 28 kb between the two ITRs of vectors to facilitate efficient packaging into virions.

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

5' ITR: 5' inverted terminal repeat. See description for 3’ ITR.

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

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