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

Boulikas: An overview on gene therapy
Figure 4. Purified recombinant AAV-mediated lacZ gene transfer to the muscle in adult mice sustains a high level of expression and
is inflammation-free. Purified AAVlacZ (1x10 9 genomes in 25 µl) was injected into the tibialis anterior of 5-week-old C57BL/6 mice
and tissue was harvested at days 3 (c), 17 (d), 30 (e), 64 (f), and 180 (g, h) post-injection and analyzed by X-gal histochemistry. Samples
of AAVlacZ (1x10 9 genomes in 25 µl) were also supplemented with an E2a mutant adenovirus dl802 (5x10 10 A 260 particles) just prior to
injection and tissue was harvested at days 3 (a) and 17 (b) post-injection. Magnification: a-g, X10; h, X5.
Purified AAVlacZ (175 µl , 1x10 12 genomes/ml) was injected into the tibialis anterior of a male rhesus monkey. Biopsies were taken
14 days post-injection and frozen sections were cut and stained for β-galactosidase activity (i and j); magnification: I, X5; j, X10.
k and l: rAAV vector expressing human β-glucoronidase was injected into the tibialis anterior of 5-week-old C57BL/6 mice (1x10 9
genomes in 25 µl). 30 days post-injection the muscle was harvested and frozen sections were cut and stained for β-glucoronidase. From
Fisher KJ, Jooss K, Alston J, Yang Y, Haecker SE, High K, Pathak R, Raper SE, Wilson JM (1997) Recombinant adeno-associated virus
for muscle directed gene therapy. Nat Med 3, 306-312. Reproduced with the kind permission of the authors and Nature America, Inc.
AAV-mediated delivery of the lacZ gene by direct
injection to brain tumors which were induced from human
glioma cells in nude mice showed that 30-40% of the cells
along the needle track expressed β-galactosidase;
subsequent delivery of the HSV-tk/IL-2 genes to these
tumors with AAV and administration of GCV to the
animals for 6 days resulted in a 35-fold reduction in the
mean volume of tumors compared with controls by a
significant contribution from the bystander effect (Okada
et al, 1996).
A phase I clinical trial for CF is being conducted at
Johns Hopkins Hospital using AAV (see Kearns et al,
1996, and protocols #165, 166 in Appendix 1).
V. Herpes Simplex Virus-1 (HSV-1)
and miniviral vectors
HSV-1 has a capacity of inserting up to 30 kb of
exogenous DNA which is a clear advantage over the
adenovirus (up to 7.5 kb of exogenous DNA). High titer
viral stocks can be prepared from HSV-1. HSV-1 also
displays a wide range of host cells and can infect
nonreplicating cells such as neuron cells in which the
vectors can be maintained indefinitely in a latent state.
However, infection with HSV-1 is cytotoxic to cells
because of residual viral proteins produced by the virus.
Strategies to circumvent this drawback led to the
development of viral vectors with a very large capacity for
insertion (almost as large as the size of the virus) which
depend on defective helper virus for replication and
packaging into infectious virions (see below). A miniviral
vector can combine the advantage of cloning the gene in
bacterial plasmids, the high efficiency of virus-mediated
18
gene transfer, and the possibility to transfer large genomic
DNA fragments including far upstream, downstream and
intronic regulatory elements.
The HSV-1 genome is a 152 kb double-stranded DNA
containing three origins of replication and encoding at
least 72 unique proteins; it consists of a unique long
segment replicated from oriL and two repeats flanking the
unique segment each replicated from oriS. Spaete and
Frenkel (1982) have constructed plasmids containing the
lytic viral origin of replication, foreign DNA inserts, and
the terminal packaging signal sequences; in the presence
of a wild-type helper virus such an amplicon was
amplified into multimeric tandemly-repeated forms of the
original vector by rolling-circle replication and was
packaged into infectious HSV virions (Spaete and Frenkel,
1982). However, the helper virus caused death of the
infected cells due to lytic replication and this system is not
amenable to gene therapy.
To circumvent this bottleneck two strategies have been
developed leading to replication-defective helper HSV: (i)
a temperature-sensitive system permitted production of
virion stocks at 31 o C whereas infection of cells at 37 o C
caused inactivation of the helper virus which was
incapable of entering the lytic cycle and allowed delivery
of the miniviral vector to the target cell without causing its
death. (ii) In a different system, the immediately early
gene IE3 was deleted from the helper virus; IE3 encodes
for a protein (ICP4) essential for early and late viral gene
expression and replication; the helper cell line used for
packaging had a genomic insertion of the IE3 gene of
HSV which was functionally expressed allowing for
complementation and for lytic infection using the IE3-