Gene Delivery and Gene Therapy for Alzheimer’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 86

replication. The ITRs fl ank the two viral genes: rep (replication),
which encodes four nonstructural proteins involved in genome
replication and packing into the nuclear capsid [ 8 ], and cap (cap-
sid), which encodes three structural proteins (virion protein VP1,
VP2, VP3) forming the capsid. The ITRs are the only cis elements
required for genome replication, packaging, and integration into
the capsid [ 9 , 10 ]. Recombinant AAV (rAAV) can be produced by
replacing the rep and cap genes with a promoter and therapeutic
gene of interest and supplying rep and cap genes in trans from a
separate plasmid lacking ITRs.
AAV is extremely promising for use as a vector for gene therapy
in the central nervous system (CNS) for several reasons. Unlike the
larger 26-45 kb adenovirus, it can infect both dividing and quies-
cent cells after a single delivery without causing pathogenicity,
immune reactions, or toxicity, leading to long-term transgene
expression [ 11 ] . Moreover, AAV shows a strong preference for
neuronal transduction [ 12 ]. Modern rAAV vectors have 96 % of
the viral genome removed from the vector; because only the two
ITRs remain, no de novo viral protein synthesis can occur after
transduction. AAV is also favorable because it has no association
with any known etiologies and unlike adenovirus, it has a very low
incidence of antigen-specifi c immunity [ 13 ].
AAV's site-specifi c genomic integration, which could reduce
the risk of insertional mutagenesis, occurs through nonhomolo-
gous recombination specifi cally in the human genome at chromo-
some 19q13.4 via AAV Rep proteins [ 14 - 16 ]. rAAV can integrate
into cultured cells at chromosome 19 if Rep proteins are supplied
in trans [ 17 ]. After AAV infection, the AAV genome enters a non-
productive, latent, non-progeny-producing state in which it exists
as a provirus integrated into the chromosomal DNA of the host
cell [ 18 ]. The potential for unwanted vector spread is limited by
the requirement of helper virus functions for a productive AAV
lytic cycle.
Because of the large number of AAV serotypes, transduced
AAV genomes confer long-term expression in a variety of cell types
and tissues, including the retina [ 19 ], muscle [ 20 , 21 ], liver [ 22 ],
and CNS [ 23 - 25 ]. Long-term expression (greater than 1.5 years)
has also been demonstrated in a number of animals, including
murine, hamster, and canine [ 20 , 26 , 27 ].

Many innovations have been made in rAAV vectorology to allow
increased specifi city, effi ciency, and spread of gene transfer, includ-
ing hybrid serotypes, rationally designed capsids, split vectors, and
specifi c promoter/enhancer additions. rAAV transduction effi -
ciency can be between 20 and thousands of vector particles per
transducing unit, although this varies depending on cell type and
differences in infectivity assays [ 28 ].

1.2 Strategies
to Increase Effi ciency
and Specifi city
of Gene Transfer: AAV
Vectorology