a) Introduction of recombinant adenovirus vector system
Since wild-type adenovirus is associated with a wide range of illnesses and enlists a variety of immune responses, so recombinant replication deficiency virus has been an attractive vector for gene therapy . To date, there have been many different generations of adenovirus vectors, differing in the extent to which the genome from wild-type adenovirus is attenuated, among them, most studies involving adenovirus vectors utilize the simple E1-deleted, first-generation adenovirus vector. Due to the induction of strong innate and adaptive immune responses and limited the duration of transgene expression of first-generation adenovirus vector, researchers have further developed the second-generation adenovirus vectors by deleting the E2 or E4 coding sequences, resulting in some beneficial effects of reducing the expression of viral proteins and vector-directed immune responses. However, because of their limited evidence of enhanced efficacy over first generation vectors, the third-generation adenovirus vectors have been designed by deletion of all viral protein coding sequences, which can only be propagated in the presence of a second virus that provides all of the replication and packaging functions in trans, for which the third-generation adenovirus vectors are also known as fully-deleted, gutted, or helper-dependent Ad (hdAd) vectors .
Traditionally, recombinant adenovirus vectors used in gene delivery were prepared with a plasmid containing the transgene flanked by inverted terminal repeats (ITRs), co-transfected with packaging plasmid pAd-BHGlox(delta)E1, E3. Once packaged into a E1-complementing cell line, such as QBI 293A cells, recombinant viral will be easily propagated.
b) Recombinant adenovirus system
The current method of the recombinant adenovirus production is based on Adeasy and Ad.MAX™ system. It involves the co-transfection of 2 plasmids into QBI-293A cells as shown in Figure 3.
1. pAd-EF1-MCS-CMV-EGFP: an adenovirus ITR-containing plasmid carrying multiple clone sites, which can be cloned into a transgene;
2. pAd-BHGlox(delta)E1, E3: a packaging plasmid.