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

On the contrary, the payload capacity of recombinant
AAV, which has been deprived of its viral genes and bears
only the ITRs is in the order of 4.5-4.7 kb; this means that
a cDNA up to this size can be inserted into a rAAV; for
example the size of the CFTR cDNA is 4.5 kb and thus,
the combined length of the promoter that drives CFTR
expression and ITRs needs to be kept under 500 bp (Dong
et al, 1996).
Similar results were reported by Dong et al (1996) who
have estimated that the optimal size of AAV vector is
between 4.1 and 4.9 kb; the packaging efficiencies were
sharply reduced above 5.2 kb and below 4.1 kb; two
copies of the vector were packaged into each virion when
vectors of 2.2-2.5 kb were provided.
C. Integration of wtAAV but not of rAAV
is site-specific
Wild-type AAV is able to undergo targeted integration
on chromosome 19 after infection in 15 out of 22 clones
examined (Kotin et al, 1990, 1992). Of 51 integrations
examined by fluorescence in situ hybridization (FISH) 48
(94%) were to chromosome 19 after infection of IB3-1
bronchial epithelial cells with wild-type AAV (Kearns et
al, 1996). Site-specific integration has been reported for
other viruses including avian leukosis virus (ALV)
integrating adjacent to cellular oncogenes in tumors;
however, the mechanism of ALV integration involves a
process of selection of cells able to form tumors by
overexpression of the oncogene due to virus integration
rather than exclusive integration of the ALV at unique
sites of the genome (Hayward et al, 1981). RSV also
appears to be integrated at a limited number of sites (Shih
et al, 1988). Adenovirus integration, a more rare event
compared to the majority of episomal molecules, may also
occur at a number of preferred sites (Jessberger et al,
1989). A larger number of recombinase molecules than
those known today may be present in mammalian cell
nuclei and promote site-specific integration and
recombination events.
Although the human wild-type AAV (wtAAV) is
unique in its ability to target viral integration to a specific
site on chromosome 19, the recombinant AAV (rAAV)
vectors have lost the site-specific integration and targeting
ability; furthermore, rAAVs have incapacitated ability to
integrate, and can be found as episomes. When wtAAV-2
was used to infect IB3-1 bronchial epithelial cells all
metaphase spreads examined by fluorescence in situ
hybridization (FISH) had integrated copies and 94% of the
integrations were to chromosome 19; furthermore, 36 of
56 metaphase spreads had a single copy of wtAAV
integrated and 20 of 56 showed two sites within
chromosome 19 (Kearns et al, 1996). On the contrary,
when a recombinant AAV containing the CFTR cDNA
was used to infect the same cells, examination of 67
Gene Therapy and Molecular Biology Vol 1, page 15
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metaphase chromosome spreads identified four
integrations (only 6% of total) to different chromosomes.
No integration was to chromosome 19. When these studies
were repeated on the A35 epithelial cell line selected for
stable CFTR expression, the episomal AAV-CFTR
sequences were abundant in the low molecular weight
DNA fraction (Kearns et al, 1996).
Yang et al (1997) have cloned over 40 AAV and
rAAV integration junctions to determine the terminalrepeat
sequences that mediate integration. These studies
have shown that in both immortalized and normal diploid
human cells, wt AAV targeted integration to chromosome
19 in head-to-tail tandem arrays; the majority of the
junction sequences were involving incomplete copies of
the AAV inverted terminal repeats (ITRs); inversions of
genomic and/or viral DNA sequences at the wt integration
site took place. The viral integration event was found to be
mediated by terminal repeat hairpin structures and cellular
recombination pathways. In contrast, rAAV provirus
integrated on chromosome 2 and at the same locus in two
independent cell lines, in both the flip and flop
orientations; genomic rearrangements took place at the
integration site of rAAV, mainly involving deletions
and/or rearrangement-translocations.
Similar data were reported by Rutledge and Russell
(1997): recombinant AAV vectors were found to be
integrated by nonhomologous recombination as singlecopy
proviruses in HeLa cells and at random chromosomal
locations; the recombination junctions were scattered
throughout the vector terminal repeats with no apparent
site specificity; the flanking HeLa DNA at integration sites
was not homologous to AAV or to the site-specific
integration locus of wild-type AAV. Furthermore, vector
proviruses with nearly intact terminal repeats were excised
from the genomic HeLa DNA and were amplified after
infection of cells with wild-type AAV and adenovirus.
The integration patterns of four recombinant AAV-2
genomes in individual clonal isolates of the human
nasopharyngeal carcinoma cell line (KB) were different;
the difference between the recombinant AAV-2 genomes
were in the combinations of the genes for resistance to
tetracycline, to neomycin, to ampicillin, with the genes for
AAV replication, and the AAV capsid genes. None of the
KB cell clones examined had the proviral genome
covalently linked to the specific-site of integration of the
wt AAV on chromosome 19 (Ponnazhagan et al, 1997a,b).
D. Drawbacks of AAV in gene therapy and
their remedy
Gene transfer with AAV vectors has typically been
low. Difficulties in generating recombinant virions on a
large scale sufficient for preclinical and clinical trials and
in obtaining high-titer virus stocks after the initial
transfection into producer cells is a limiting factor for the