Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 227

4. When a latently infected cell is superinfected with a helper virus, such as adenovirus or
HSV, the AAV gene expression program is activated, leading to the AAV rep-mediated res-
cue (i.e., excision) of the provirus DNA from the host cell chromosome, followed by repli-
cation and packaging of the viral genome.
5. The newly assembled virions are released upon the helper virus-induced cell lysis. The
induction of the lytic phase of the AAV life cycle from a stably integrated provirus can also
occur in the absence of a helper virus, though with a lower efficiency, when the host cell is
subjected to metabolic inhibitors, DNA damaging agents, or genotoxic compounds.
5.3.3.2 AAV Tropisms
AAV2 is the most widely used AAV serotype for in vitro and in vivo gene delivery.
Every year there are some new serotypes discovered, and many other serotypes of AAV
have been isolated. Among them, AAV1, AAV2, AAV5, as well as AAV7, AAV8 ,and
AAV9, as well as others, have been used for gene delivery studies. The main complica-
tion is choosing an appropriate or best among these serotypes of AAV, for a particular
study model. The differences in efficacy of gene delivery are unknown in spite of the
numerous studies in various models. It is generally believed that differences in animal
species involved in study protocols, end-point readout, tissue or organs, as well as the
procedures used to prepare those recombinant viruses, as well as other considerations,
could contribute to this. For comparison of efficacy, it is better to use several rAAVs
carrying a reporter gene, such as GFP or LacZ , to decide which serotype and procedure
is appropriate for a particular study.
5.3.4 Design and Construction of Adeno-Associated Viral Vectors
5.3.4.1 A. Design
The principles behind the construction of an AAV vector [97-99] are generally based
upon the modification of molecular clones by substituting the AAV-coding sequence
with foreign DNA to generate a vector plasmid. Only the cis -acting ITR sequences
must be kept intact during this type of modification. The vector plasmid is introduced
into producer cells that are also rendered accommodating by an appropriate helper
virus, such as adenovirus.
To accomplish replication and encapsidation of the vector genome into AAV par-
ticles, the vector plasmid must be complemented for the transacting AAV rep and cap
functions; these transacting AAV rep and cap functions were deleted in construction
of the vector plasmid. Vector particles of AAV can be purified and concentrated from
lysates of such producer cells. About 5 kb of DNA can be packaged in an AAV vec-
tor particle. This places constraints on the expression of very large cDNAs and may
also restrict the ability to include regulatory control sequences in the vector. The capsid
also interacts with the AAV receptor and coreceptors, and thus mediates cell entry. The
capsid may also induce humoral immune responses, so it has limited application for
delivery.
In spite of all these limitations, the ITR can act as a transcription promoter
[100,101] but does not interfere with other promoters. These tissue-specific promoters