Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 27

virions are finally released upon cell lysis. Since AAVs do not possess
any lytic activity per se, release of newly assembled virions relies on
the lytic activity of the helper virus.

1.2 AAV as a Vector
for Gene Therapy

Recombinant AAVs (rAAV) are generated by replacement of the
entire rep and cap ORFs by an expression cassette containing
the gene of interest and specific regulatory elements [ 40 ]. Since
the ITRs are the only viral sequences retained in the rAAV genome,
the AAV rep and cap genes and helper functions are therefore pro-
vided in trans to ensure amplification of the rAAV genome and
production of rAAV infectious particles [ 40 ]. Importantly, rAAVs
are unable to replicate their genome on their own and to site spe-
cifically integrate into the host genome as they are deleted for the
rep gene. The majority of AAV vector genomes is nonintegrating
and persists predominantly as circular monomeric and concate-
meric episomal forms [ 41 ], which are likely responsible for long-
term transgene expression [ 41 , 42 ].
Several limitations for the use of AAV vectors as gene delivery
vehicles led to the search and development of better vectors. One
of the major limitations of rAAV vectors is the limited packaging
capacity, which cannot exceed 4.5 kb. Dual vector approaches
based on the unique ability of AAV to form concatemers are cur-
rently used to increase the packaging capacities of rAAV vectors.
This approach consists of splitting different elements of the trans-
gene cassette or the gene by itself into two vectors; transgene
expression is therefore restored only upon head to tail recombina-
tion [ 43 , 44 ]. A further limitation of current use of AAV vectors
concerns a number of rate limiting steps in AAV gene transduction
including intracellular trafficking to the nucleus, viral uncoating,
vector genome unstability, and second-strand synthesis. To bypass
the requirement for double-strand synthesis, self-complementary
or double-stranded vectors have been developed resulting in a
transcriptional-competent double-stranded DNA template [ 45 ].
However, the main limitation of using these scAAV vectors resides
in the packaging capacity, which is reduced by approximately
twofold.
Despite the broad cell tropism of AAV-2, certain cell type can-
not be efficiently infected by AAV2. Taking advantage of the natu-
ral diversity of other serotypes, pseudotyping approaches have
been developed consisting of cross-packaging the AAV2 genome
into capsids derived from different serotypes [ 46 ]. These methods
not only improve transduction of certain cell type but also circum-
vent the existing problem of neutralizing antibodies. However,
some tissues remain refractory to transduction by natural AAV
serotypes. Therefore, strategies to modify existing AAV capsids
and modulate AAV tropism have emerged including generation of
chimeric or mosaic capsids, insertion of ligands into different
regions of the capsids, library selection and directed evolution of
the AAV capsid [ 47 ].