Targeted Gene Therapy for Ischemic Stroke - Gene Delivery and Therapy for Neurological Disorders - page 192

control the therapeutic gene expression for a safe clinical application.
Examples of inducible promoters that have been used to control
gene expression include the tetracycline operon, RU-486, and
edyasone [ 12 - 20 ]. Limiting gene expression to hypoxia conditions
is ideal for the treatment of ischemic tissue.
HIF-1 is a protein that accumulates in many tissues under
hypoxic conditions. This protein is a heterodimeric basic helix-
loop-helix protein with a labile ʱ subunit (HIF-1 ʱ ) and a ʲ subunit.

The subunit is constitutively expressed in tissues in normal and

hypoxic conditions [ 21 , 22 ]. HIF-1

ʲ

protein is rapidly degraded via
ubiquitination and proteasomal digestion, a process mediated by a
200-amino acid oxygen-dependent degradation domain [ 23 ]. This
subunit is stabilized in hypoxic tissues and binds with the

ʱ

subunit
to form functional HIF-1 protein that activates transcription of sev-
eral genes, including erythropoietin (Epo) and vascular endothelial
growth factor (VEGF) in hypoxic cells [ 24 ]. Our study showed that
HIF-1 is greatly increased in the ischemic brain one day after MCAO
[ 25 ]. Therefore, HIF-1 acts as an early sensor of hypoxia and may
trigger gene expression in ischemic tissues. We also demonstrated
the feasibility of controlling AAV-mediated VEGF gene expression
in a hypoxia-inducible manner by using nine copies of HRE isolated
from Epo gene enhancer and a minimum SV40 promoter [ 4 , 26 ].
AAV is an ideal vector for delivering genes into the brain
because it effectively infects neurons and astrocytes [ 5 , 27 , 28 ].
More than 90 % of the AAV genomes remain episomal in infected
cells [ 29 ], and thus AAV infection is unlikely to cause insertional
mutation and tumor formation. The vector itself has not been
associated with toxicity or any infl ammatory response (except for
the generation of neutralizing antibodies that may limit readminis-
tration) [ 30 ]. rAAV-transferred BDNF or GDNF shows neuropro-
tection in both Huntington's [ 31 ] and Parkinson's [ 32 ] animal
models in vivo. There are many AAV serotypes available for pack-
aging AAV vectors. Different AAV serotypes infect different tissues
and cells with varying effi ciencies, providing opportunities to select
one best suited for a specifi c purpose.
Because it is diffi cult to effi ciently deliver viral vectors across
the BBB, the delivery of therapeutic genes to the brain remains a
major challenge. Wild-type AAV has a single-stranded genome.
AAV with a single-stranded genome (ssAAV) requires host-cell
synthesis of the complementary strand for transduction. When the
genome is half wild-type size, AAV capsid can package either two
copies or dimeric inverted repeat DNA molecules. Dimeric, or self-
complementary molecules (scAAV), spontaneously reanneal, alle-
viating the requirement for host-cell DNA synthesis, which results
in rapid and higher levels of transgene expression than ssAAV [ 33 ].
Recombinant AAV packaged in an AAV9 capsid can effectively pass
through the BBB [ 34 , 35 ], but only scAAV9 (not ssAAV9) robustly
mediates transgene expression in the adult brain after intravenous

ʲ