11viral and host factors. These genes are often removed or manipulated to both make
room for transgenes as well as decrease the ability of the virus to replicate after
transduction into the host cell [ 86 – 88 ]. While there are over 50 serotypes of adeno-
virus, the most commonly used and best understood is serotype 5, often referred to
as Human adenovirus serotype 5 (HAdV-5) [ 89 ]. These viruses, unlike retroviruses,
have no endogenous integration machinery and do not incorporate into host genomes
at high frequency, instead remaining as episomal elements [ 89 ]. Their episomal
nature means that they have a much lower mutagenic potential than retroviruses.
Naturally, the HAdV-5 vector has an affinity for transduction in hepatocytes, which
is a benefit for delivery of transgenes to the liver, but a downside if other cell targets
are desired [ 89 , 90 ]. Additionally, adenoviral vectors have been shown to be highly
immunogenic, due to natural exposure to adenoviral particles that most humans
experience early on in life [ 89 , 90 ].
1.4.1 Modifications and Implementation of Adenoviral Vectors
One of the first applications of HdAdV-5 for gene therapy was by Jaffe et al. who
deleted the E1 and E3 viral genes to inhibit viral replication and make room for
the human α1-antitrypsin gene and a β-galactosidase gene (as a marker of viral
transduction). After intraportal injection into rats, the group found that
α1-antitrypsin was detectable in serum for up to 4 weeks, demonstrating the power
of modified adenoviral vectors for gene therapy [ 86 , 91 ]. Shortly after, the same
group showed the efficacious use of the HAdV-5 vector without E1/E3 genes to
transfer human Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
genes into the respiratory epithelium of rats, demonstrating the potential for such
vectors to treat Cystic Fibrosis [ 92 ].
Further removal of essential viral genes has produced vectors with transgenesflanked by inverted terminal repeats (ITRs, necessary for packaging the genome
into the vector) referred to as gutless adenoviral vectors, with the viral genes needed
for production supplied by the cell line used to manufacture the virus [ 93 , 94 ].
“Gutless” vectors have been used to introduce DNA into human induced pluripotent
stem cells (iPSCs) and embryonic stem cells (ESCs) by homologous recombination
[ 88 , 95 , 96 ]. For example, such vectors have been used to repair genes involved in
laminopathy, muscular dystrophy, and hemophilia B [ 96 – 98 ]. Given the large
genome size of adenoviruses, these vectors are ideal delivery systems for genes that
are too large for other viral vectors.
In addition to solely delivering DNA to replace or complement ineffective/mutated genes, groups have also delivered nucleases and recombinases that stim-
ulate recombination between the donor DNA and the host genome [ 89 , 99 – 101 ].
As discussed with lentiviral vectors, zinc-finger nucleases, TALENs, and CRISPR-
Cas systems have been similarly delivered with adenoviral vectors [ 100 , 101 ]. For
instance, Perez et al. used zinc-finger nucleases encoded in a viral vector to dis-
1 Viral Vectors, Engineered Cells and the CRISPR Revolution