Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 225

Table 5.4 Advantages and Drawbacks of AAV

Advantages of AAV Drawbacks of AAV

The viruses have an apparent lack of pathogenicity
Viruses can also infect nondividing cells. They have
the ability to stably integrate into the host cell genome
at a specific site in the human chromosome
(designated AAVS-1)
The AAV genome integrates most frequently into the
site mentioned, while random incorporations into the
genome take place with a negligible frequency
Development of adeno-associated viral vectors as gene
therapy vectors has eliminated this integrative capacity
by removal of the rep and cap from the DNA of the
vector
AAV-based gene therapy vectors form episomal
concatamers in the host cell nucleus. In nondividing
cells, these concatamers remain intact for the life of
the host cell. In dividing cells, AAV DNA is lost
through cell division, because the episomal DNA is
not replicated along with the host cell DNA
Random integration of AAV DNA into the host
genome is low but detectable
AAVs have very low immunogenicity, seemingly
restricted to generation of neutralizing antibodies.
Cytotoxic response is not clearly defined

Adeno-associated viral vectors have
relatively limited cloning capacity,
and most therapeutic genes require
the complete replacement of the
virus's 4.8 kb genome
Large genes are not suitable for use
in a standard AAV vector
To overcome the coding capacity
limitation, the AAV ITRs of two
genomes can anneal to form
head-to-tail concatamers, almost
doubling the capacity of the vector.
Insertion of splice sites allows for
the removal of the ITRs from the
transcript
The associated neutralizing activity
limits the usefulness of the most
commonly used serotype AAV-2 in
certain applications

5.3.3 Structure and Biology of Adeno-Associated Virus
5.3.3.1 AAV Structure
5.3.3.1.1 Genome, Transcriptome, and Proteome

1. The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either “posi-
tive sensed” (same base sequence as mRNA) or “negative sensed” (base sequence comple-
mentary to that of mRNA), and about 4.7 kb long.
2. ITRs at both ends of the DNA strand and two open reading frames (ORFs): rep and cap.
The former is built up of four overlapping genes encoding rep proteins, which
are required for the AAV life cycle, and the latter contains overlapping nucleotide
sequences of capsid proteins (VP1, VP2, and VP3). Interaction of these proteins
forms a capsid with an icosahedral symmetry. These capsid proteins assemble into a
near-spherical protein shell of 60 subunits. The virus does not encode a polymerase
and therefore relies on cellular polymerases for genome replication.
The ITRs flank the two viral genes— rep (replication) and cap (capsid)—encoding
nonstructural and structural proteins, respectively, and they comprise 145 bases each.