Adenoviral genes	Function
Early genes: E1A, E1B, E2, E3, E4	Transcription, replication, host immune suppression, inhibition of host cell apoptosis.
Delayed early genes: pIX, IVa2	Packaging
Major late gene (L)	Assembly

Table 5-1.4.1: Different types of adenoviral genes and their function.

Construction of Adenoviral vectors
First generation adenoviral vectors were replication deficient , lacking the essential E1A and E1B genes and often the non-essential gene E3 and were called ‘E1 replacement vectors'. They had a maximum capacity of about 7 kb and were propagated in the cell lines transfected with DNA containing E1 genes e.g. human embryonic kidney line 293 (HEK 293).
Drawback

• These vectors caused cytotoxic effect due to low-level expression of the viral gene products, and chances of recombination between the vector and the integrated portion of the genome, resulting in the recovery of replication-competent viruses.
  
  

  Higher-capacity vectors lacking the E2 or E4 regions in addition to E1 and E3 provide a maximum cloning capacity of about 10 kb but still allow low level of transgene expression. These must be propagated on complementary cell lines providing multiple functions. The use of E1/E4 deletions is a sound strategy as the E4 gene is responsible for many of the immunological effects of the virus.

  To overcome the above limitations, an alternative strategy employs insertion of ‘stuffer DNA' into the nonessential E3 gene as part of the vector backbone so to maintain optimum vector size. Helper dependent adenoviral vectors (HDAd) are favoured for in vivo gene transfer due to deletion of all viral coding sequences.

Advantages of HDAd

• Large cloning capacity (up to 37 kb)
• High transduction efficiency
• Long term transgene expression
• Lack of immune response and cytotoxicity.