01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

Boulikas: An overview on gene therapy
Figure 3. Comparison of the persistence of dystrophin expression and adenoviral genomes in immunosuppressed versus
immunocompetent mdx mice. Shown are combined dystrophin immunostaining and in situ PCR in tibialis anterior muscles of mdx mice
at 10 days (A and C) and 60 days (B and D) postinjection. In A and B, FK506 was used as an immunosuppressant, whereas in C and D
no immunosuppression was employed. At 10 days there was no significant difference in adenovirus positive nuclei (arrows) fibers
between the immunosuppressed and the immunocompetent groups. At 60 days, however, there was a dramatic decline in the number of
positive nuclei in the immunocompetent muscle. Magnification 650X. From Zhao JE, Lochumuller H, Nalbantoglu J, Allen C, Prescott
S, Massie B, Karpati G (1997) Study of adenovirus-mediated dystrophin minigene transfer to skeletal muscle by combined microscopic
display of adenoviral DNA and dystrophin. Hum Gene Ther 8, 1565-1573. With kind permission of the authors (George Karpati,
Montreal Neurological Institute, Canada) and Mary Ann Liebert, Inc.
IV. Gene delivery with Adeno-
Associated Virus (AAV)
A. Replication of AAV and rAAV: the role
of the inverted terminal repeats
AAVs are replication-defective parvoviruses, not
associated with any human disease (nonpathogenic),
requiring cotransfection with a helper virus to produce
infectious virus particles; they can replicate in cell culture
only in the presence of coinfection with adenovirus or
herpes virus. Five serotypes of distinct AAV isolates have
been recovered from human and other primates. AAV
infections in humans are asymptomatic acquired with
other viral infections such as adenovirus or HSV
infections; 80-90% of adults are seropositive for
antibodies against AAV (for references see Clark et al,
1995; Berns and Linden, 1995).
The replication of the AAV is dependent on two copies
of a 145-bp inverted terminal repeat (ITR) sequence that
flanks the AAV genome which is the primary cis-acting
element required for productive infection and the
generation of recombinant AAV (rAAV) vectors.
In the absence of helper virus, the AAV particle can
penetrate cells and find its way to the cell nucleus where
the linear genome is uncoated and becomes integrated at a
specific site on chromosome 19q13.3; several copies of
AAV may integrate in tandem arrays. Thus, the AAV
establishes a latent infection; the integrated viral genome
can be activated and rescued by superinfection with helper
virus (either adenovirus or any type of herpes virus).
Inverted repeats at the ends of the viral DNA serve for the
integration appearing near the junctions with cellular DNA
sequences (Bohenzky et al, 1988).
Adenovirus establishes foci called replication centers
within the nucleus, where adenoviral replication and
transcription occur; AAV was colocalized with the
adenovirus replication centers using in situ hybridization
and immunocytochemistry; AAV may, thus, utilize
adenovirus and cellular proteins for its own replication;
the rAAV genome was faintly detectable in a perinuclear
distribution after successfully entering the cell; however,
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rAAV was mobilized to replication centers when the cell
was subsequently infected with adenovirus (Weitzman et
al, 1996).
Xiao et al (1997) have engineered the pDD-2 plasmid
containing two copies of the D element, a unique sequence
adjacent to the AAV nicking site, flanking a single ITR (a
total of only 165 bp of AAV sequence); this modified
hairpin was sufficient to sustain replication of the plasmid
vector when Rep and adenovirus helper functions were
supplied in trans. This plasmid has a significant prospect
in gene transfer because is replicated more efficiently than
infectious AAV clones; as a prelude to its replication the
input circular plasmid was converted into a linear substrate
by resolution of the AAV terminal repeat through a
Holliday-like structure, a process most likely mediated by
host factors. Linear monomer, dimer, and other highermolecular-weight
replicative intermediates were generated
during the replication of pDD-2, a feature characteristic of
AAV replication. The replicative intermediates of this
plasmid substrate were competent for AAV DNA
replication, encapsidation, infection, integration, and
subsequent rescue from the chromosome when
superinfected with Ad and wild-type AAV (Xiao et al,
1997). The elucidation of the important role of this 165-bp
ITR sequence for AAV replication and the entire life cycle
invigorates the important role of inverted repeats at the
origin of replication not only of viruses but also of cellular
origins of replication (Boulikas, 1996e).
B. Packaging capabilities of AAVs
AAVs posses a 4.7 kb single-stranded DNA genome.
Hermonat et al (1997) have examined the maximum
amount of DNA which can be inserted into the wild-type
AAV genome without compromising packaging into an
infectious virus particle; the maximum effective packaging
capacity of AAV, examined as increments of 100 bp
ligated at map unit 96 of AAV, is approximately 900 bp
larger than wild type. Thus, wtAAV therapy vectors can
be generated carrying a foreign gene of 900 bp or less with
the advantages of wtAAV such as the ease in which high
titers of infectious virus can be generated and the ability to
specifically integrate in chromosome 19.