Chimeric Adenovirus Vector Construction Service
Adenovirus (Ad) vectors are potent gene delivery vectors that cause mucosal and systemic immune responses. However, the presence of circulating antibodies to Ad capsid proteins is a barrier to gene therapy. By modifying the Ad fibrin, for example, by constructing a chimeric Ad vector, the properties of the fibrin can be altered, thereby increasing the efficiency of the vector in gene therapy. Creative Biolabs is a biotechnology company specializing in custom Ad vector production services. We offer a wide range of services for the construction of chimeric Ad vectors.
Introduction of Chimeric Ad Vector
Prototype Ad vectors for gene therapy have been developed based on human adenovirus C (HAdV-C), such as serotype 5 (HAdV-5). Owing to frequent exposure to these pathogens, neutralizing antibodies against the HAdV-C species in human populations are usually highly prevalent. This fact may limit the effectiveness of gene therapy vectors based on HAdV-C serotypes. Therefore, the successful use of any Ad vector depends on the use of serotypes, which cannot be neutralized by circulating antibodies. In view of the above, it is feasible to construct chimeric Ad vector in which most of the structural proteins are derived from Ads of unrelated serotypes. This retains most of the protein-protein interactions associated with capsid assembly. It is indicated that the strategy has a wider applicability.
Figure 1. Schematic representation of Ad structure. (Raty, 2008)
Chimeric Ad Vector Construction
Chimeric pseudo-vectors are commonly used to modify Ad tropism by replacing fibrin. The overall method towards constructing a chimeric virus is firstly to assemble the complete E1-deletion-virus DNA into a single plasmid, digest the plasmid DNA with an appropriate restriction enzyme to release the viral DNA ends, and transfect the DNA into a cell line such as HEK 293 to determine whether viable chimeric Ad can be rescued. Creative Biolabs has successfully constructed a chimeric Ad vector to construct all recombinant Ads by assembling a complete recombinant Ad genome in a single plasmid, which is flanked by a PacI site for the release of recombinant viral genomes from the plasmid backbone. Then, it is transfected into 293 cells to rescue the recombinant adenovirus. Most of the capsid proteins of this chimeric vector are derived from one adenovirus species and the flanking E1 and E4 regions are from another.
Figure 2. Construction of chimeric Ad vectors. (A map of the genome of the vector C7eGFP with the locations of the principal Ad protein-coding regions is shown). (Roy, 2005)
Taking the most widely used group B (Ad3) and group C (Ad5) Ads as example, the chimeric fiber cDNA containing the head domain of Ad3 fiber fused with the tail and shaft of Ad5 fiber was integrated into the genome of an Ad vector, where E1 and E3 were deleted to encode beta-galactosidase to produce an Ad particle which contains a chimeric fibrin. Transduction experiments with fibrin competitors showed that the altered receptor tropism of chimeric fibrin vector is comparable with parental vector. Vector containing Ad3 fiber is more efficient than Ad5 fiber in transducing three cell lines (THP-1, MRC-5 and FaDu). These results suggest that the exchange of fiber head domains is a feasible method to produce Ad vectors with cell type selective transduction characteristics. Therefore, this method of constructing chimeric Ad vectors can be extended to use ligands for a series of different cell receptors in order to target gene transfer to specific cell types at the transduction level.
Advantages
Chimeric Ad vectors for gene therapy are mainly used to increase the host range, expand the capacity and the titer of transgenic gene production, and combine gene elements to prolong the time of gene expression. At Creative Biolabs, we can provide you a series of chimeric Ad vector construction services, with the following advantages:
- Customization service: cloning scheme can be determined according to the original plasmid information provided by customers;
- Short turnaround time: the construction of viral DNA from the original plasmid can be completed in a short time;
- High cloning positive rate, reliable quality, stable packaging, rapid toxic and high titer.
If you are interested in our chimeric Ad vector construction services, please feel free to contact us or directly send us an online inquiry.
References
- Raty, J.K.; et al. (2008). Gene therapy: the first approved gene-based medicines, molecular mechanisms and clinical indications. Current molecular pharmacology. 1(1): 13-23.
- Roy, S.; et al. (2005). Use of chimeric adenoviral vectors to assess capsid neutralization determinants. Virology. 333(2): 207-214.
